Honest Protocols: SDS PAGE

So, this time we looking at the fun and active (full sarcasm intended) stage of the western blot: SDS Page Electrophoresis. This is a form of gel electrophoresis specifically for proteins. When I had to do this for the first time, I really struggled to find any practical information on how to actually do it and was so reliant on the kindness of lab members to help me out. Still took me ages to do it probably. So, I'm hoping this one will be helpful to any poor person who is trying to do this for the first time. 

The point of the SDS electrophoresis is to separate molecules like proteins according to their size. Because the proteins have a charge, an electrical current separates them out. The smaller molecules move faster and further than the heavier ones. This allows you to see what proteins are actually present in the sample. Each protein forms an isolated band which you can work out the size off.  You can do further experiments with these separated proteins -such as the western blot. 

What a SDS electrophoresis looks like. 

So down to business:

1) Draw up the excel sheet where you calculated how much of the sample you actually needed for the electrophoresis*

*The amount of protein you need for a gel electrophoresis is about 10-50 µg per sample. If you have too much protein, it won't migrate probably. So, what you need to do is dilute the samples down with some distilled water. You don't want to use the entire sample so the best way to do is work out the concentration of the sample, work out how much µl you actually want and add the required amount of distilled water to get the amount of protein down to 10-50 µg. So, for example, if your BCA tells you have 20 ug of protein and you took 10 µl of lysate for the BCA, that gives you a concentration of 2 µg/µl- which is also 2 mg/ml. So, for every 10 µl of lysate, you have 20 µg of protein. Now let’s say you want 10 µg of protein per well. But let’s say the gel well holds up to 40 µl of solution in total. You decide to make it out to 30 µl so you need 5 µl of the lysate plus 25 µl of distilled water. 

2) Hunt down the distilled water and the Laemmli buffer* from wherever the last person put it.

*The Laemmli buffer is used to give you better bands as it helps with the isolation of the proteins. It has five crucial components including a dye, but most labs have it already made for you. Fair warning, it smells AWFUL. When adding it to your water + protein mixture, you want the L buffer to be a sixth of the total volume- including the buffer. So, if the protein and the water combined are 30 µl, you can add 6 µl of the L. buffer. 

Laemmli Sample Buffer from Bio-Rad. It's purple and smells gross. 

3) Sulk at the smell of the buffer. Question your life choices.         

4) Consider the gel percentage* you will need.  A

*Gels are made out of something called polyacrylamide which gives you a matrix full of pores. The proteins move though the pores to separate out. Proteins between 25-100 kDa will need about 8% polyacrylamide but a smaller one of between 4-40 % kDa will need about 20 %. More polyacrylamide will give you decreased pore size. So larger pore sizes are better for your bigger proteins. Of course, if you are trying to investigate several proteins that have different sizes, you may have to run them all on separate gels which is just a nightmare. You can also use something called a gradient gel which is made up with a range of concentrations. 

5) Hunt down the helpfully pre-cast gels* in the cold room. 

*Pre cast gels are a luxury that some labs get. The gel is already made up for you, and it is contained within a plastic casing. It also has been created with a plastic comb which creates the wells you will need. The wells are where the samples are going to go. 

A precast gel with green comb and tape in a wrapper. 

A gel after running the SDS page. The gel itself is contained within the plastic 

and the blue is the L.buffer that has travelled down. 

6) Remove the comb and the sticky tape* from the gels.

*The sticky tape is designed to present the gel getting dehydrated or breaking in transport.  But you don't remove it, you stop the electrical current from getting to the gel. So, for the love of GOD, remove it!

7) Place gel in the tank*. Realise you are going to need to get a second gel and run two gel electrophoresis*.

*The tank is what holds the gel and has the running buffer which is going to carry the electric field. It also has two electrodes - a positive anode at the bottom and the negative cathode at the top. The proteins are going to move down towards the positive anode as they are negatively charged. The lid is also incredibly important and is going to connect the tank to your power supply. 

                                         An SDS PAGE tank with the lid. 

*Each tank holds two cassettes which holds two gels each. So, you get four in total.  You only need to use one cassette - but to seal the cassette probably to minimise leaking, you need to use two gels. You could potentially use a blank which is just a piece of plastic the same size as the gel and its plastic casing, but they are always a nightmare to hunt down. So, it's just easier to run two. 

Tank showing the cassette. The gels get clamped in front and behind of the white plastic

 and are held in place by the green. 

8) Make up a second set of samples for your second gel electrophoresis. 

9) Hunt down a power pack. Remind yourself how to set up a tank*.

Power pack. It's usually lurking on someone's lab bench. 

*Luckily these tanks are usually colour coded. Just remember: RED ELECTRODE TO RED SIDE

10) Realise you have run out of running buffer*

*Running buffer is what the electrical current is going to travel though in your SDS PAGE electrophoresis. It's made up of Tris - which keeps the pH stable, glycine which helps the proteins line up neatly before separating - think of it like them all meeting at the same starting point, and it also has SDS. This keeps the proteins unfolded and negatively charged.

11) Make up some more.  Make up extra to use in another electrophoresis in a rare exhibition of forward thinking*

* Because you use so much of it, you're better off making a 1 litre stock that is 10 X more concentrated that you need and then dilute it 10 times when you are using it. For example, take 100 ml of your stock and dilute it with 900 ml of running buffer. 

12) Fill tank with running buffer*.

*You want enough to cover the wells in the inner chamber - the space between where the two gels are clamped in the cassette, and enough to cover the bottom edge of the gel in the outer chamber. It's about a litre.

You can just see the running buffer in the outer chamber, and in the inner chamber, 

it should be filled to the top of the wells. 

13) For once, remember to wash out the wells with running buffer*

*Sometimes the wells get clogged with the gel, so you want to make sure they are empty. 

14) Scrabble blindly in the freezer and eventually hunt down the ladder *

*The ladder is a mixture of proteins with known molecular weights. It's like a size ruler. You can compare the bands that your sample produces with the bands the ladder gives you to estimate the size of the proteins you have. 

A ladder produces something like this. Each ladder comes with a guide 

telling you what size each band is.

15) Mentally brace yourself for the fun of loading* into the well.

*Loading is almost as hellish as a western blot. You are using a pipette to place small amounts of fluid in a well in the gel which is almost invisible. 

Like that. It's easier once you have the first well filled. 

16) Load 4 µl of ladder into each well. Decide to use the ladder to help you distinguish between the left and right of the gel.  Load two ladders at the left side, one at the right*

    * This will be important as it will help you remember what samples were actually in each well. The gel will be flipped around later so it's hard to remember which well was which. But if well 1 was the one next to two ladders, you can work it out. 

17) By some miracle, you manage to get the ladder into the well without missing it or stabbing the bottom of the well with the pipette tip.

18) Brace yourself for more loading.

19) Load 13ul of your prepared samples into the wells. Cry internally.

20) Mentally swear abuse at whoever is making noise in the other end of the lab.

21) Breathe for the first time in five minutes when you finish without stabbing any wells or missing any. 

22) Put the lid of the power pack on your gels. Remind yourself again that it is RED to RED. 

23) Attempt to run the power pack.

24) Scream when you see there's an ERROR alert on the powerpack.

25) Realise it's because your tank is leaking and the running buffer is no longer covering the tops of the well*.

*Leaking tanks are a rite of passage. You will spend ages trying to find the one tank in the lab that doesn't leak, think you have found it and not realise you have not got it until it's too late. 

26) Clean up the buffer that has leaked over your table and top up the tank.

27) FINALLY, the powerpack is good to go.

28) Settle on a voltage of 100 V *.

*100 V is a standard voltage to use. It's a good one to use if you haven't mucked around with the protein before. 

29) Debate whether you should run it for 1 hour or 2 hours.

30) Decide on 1 hour and you will come and check on it later.

31) Go grab a coffee and hunt down your will to live. 

32) Come back to the lab and decide it still needs a half hour.

                                     The samples should form a purple line and

                                             gradually move down the gel.

33) Hike up the voltage to 120 V.

34) Decide you can't be bothered to take off your lab coat again so settle down to wait. 

It looks a bit like this when it's finished. Either side is the ladder.

 You can only see the dye in the samples but proteins of larger sizes will be present

 further up the gel. The next stage is to do the western blot to see what exactly is present.  

 Once the gel has finished, it's time for transferring and blocking which will be explained in the next honest protocol.  Which is more hellish than the loading. To be honest, everything gets hellish from this point on. HAVE FUN!

Honest Protocols! BCA assay

 Third entry in my honest protocols series and today we are talking though the BCA assay.

Once we have collected our lysates, we need to know how much protein is actually contained with the lysate.  This is so we can actually use these samples for experiments. So, we use something called the BCA. I always hated having to do this, because I just couldn't win either way. If the assay didn't work, then my samples didn't have the protein I needed and they were useless. But if the assay worked, then I would have to do a western blot. Which was just absolute hell. But the one consolation is that it was reasonably quick to do, giving you some time to accept your fate and you get a nice purple colour when the assay works. 

The purple solutions.

The BCA measures the protein concentration using something called the Biuret reaction. In this reaction, you add a base to your protein making it alkali. Then, you add copper sulphate. Copper sulphate has copper ions that have a charge of 2+. Peptide bonds - the bonds holding amino acids together to form a protein, have nitrogen ions. These nitrogen ions react with the 2+ copper ions and make them 1+ copper ions.  By detecting how much of these single charge ions you have, you can in theory work out how much protein you have.

The best way to work out how many 1+ copper ions you have to create a colour change. This is where the BCA comes in. BCA stands for bicinchoninic acid, and it reacts with 1+ copper ions to form these great purple colour. So, the more purple you have, the more protein you have. 

What you also have is are the Bovine Serum Albumin standards -or BSA for short. These standards contain a known amount of protein, so you run the assay, and create what's called the 'standard curve'.  You can then run your actual samples and find the data on the standard curve, telling you how much protein you have in your samples. 

The BCA assay kit I used always contained a few bottles. The BCA held in an alkaline environment which was always in this big bottle, the copper solution which was in a little bottle and is bright blue and the Bovine Serum Albumin (BSA) standard which were in these tiny ampules. So, I used to remember them as the Big Bottle, the blue stuff and the tiny bottles.

The big, the tiny and the blue.

The copper sulphate solution has to be added to the BCA solution just before you use it. This is because adding copper sulphate to this solution makes a very chemically unstable solution that starts to degrade pretty quickly. So, it stops working if it gets too old. Bit ageist to be honest. 

Anyway, here's what you need to do in as much detail and as honesty as I can possibly give:

1) Hunt down the BCA box that's somewhere in the lab.

2)  Realise it is not in its usual location.

3) Panic for a moment, thinking that there's no BSA anywhere in the lab and you won't be able to run a western blot. Brighten up for a moment realising that you won't HAVE to run a western blot. Then sulkily realise that Kate at the lab bench across the room was also doing a western blot and nicked the BSA, and you no longer have an excuse to get you out of having to do the western blot.

4) Stomp across the lab, have a nice conversation with Kate and walk back with the BSA.

5) Consider how much of the big bottle you will need and how much of the blue stuff you will need to add*

*It's 0.2 ml of the blue stuff to 9.8ml of the stuff in the big bottle.

6) Realise you haven't even got your 96 well plate yet, so wander over to the store room to retrieve it.

7) Your favourite lab marker pen* ran out yesterday and you haven't replaced it so yet, so wander to annoy your favourite lab friend and wheedle a lab marker pen out of them.

*Alongside blue roll, lab marker pens are one of the most useful things ever. These pens are designed to withstand the harshest temperatures and the only way you are getting it off your lab equipment is a lot of ethanol and elbow grease. 

8) Label your 96 well plate CLEARLY*

*An unlabelled 96 well plate can ruin everything. You need to mark where your standards are going to be and where your ACTUAL samples are going to be placed, along with your controls.  

Diagrams like this are useful for planning

 where each of your standards and samples will go. 96 well plates have a lid so you can use your lab marker to actually mark the lid where everything should go. 

9) Make the BCA, put it aside and start making up your BSA standards. 

10) Realise that the standard is in this flipping glass ampule that you have to break open to access the liquid. 

Like this. The join is always weaker where that green band is

 so that's where you should aim to break it.

11) Gingerly break it open without cutting your fingers. Always a miracle. 

12) Create first standard with 30 µl* distilled water and 0 µl BSA. Do not create it directly in the 96 well plate. Make it up in E-tubes.

*µl stands for microlitre. It's a flipping tiny measurement.  

13) Create second standard with 24 µl water and 6 µl BSA. 

14) Keep going until last sample contains 0 µl water and 30 µl BSA*

    *If this sample goes purple, you have majorly screwed up. This sample should not contain any protein. 

15) Vortex all your samples for a few seconds or so. Stick your finger in the vortex when no one is looking.

 

Standard Lab vortex.

16) Pipette your standards onto your well plate. Each well should have 10 µl and you want to have at least two wells for each standard. Three is better. 

17) Panic for a minute when you forget which one of your E.tubes had which standard and there's a chance you pipetted the wrong one into the wrong well. Decide you can't have because there's still the same number of liquid in the E.tube that shouldn't have been used as all the other tubes. The only one with less liquid is the one you should be using. 

18) Realise it might have helped if you labelled the E.tubes with the coordinates of the relevant well but that no one is ever that forward thinking.   Decide you will do that next time, knowing that you will have forgotten by then.

19) Hunt down your lysates in the -80 °C freezer. 

20) Realise that it might have been useful if you had taken them out of the freezer before you started wandering around the lab, because now you need to wait for them to defrost.

21) Luckily, the samples are small enough, and shouldn't take too long. You half -heartedly flick them*

*This actually works to defrost small volumes. 

22) Your samples finally defrost so you can start pipetting them. You want 1 µl of sample and 9 µl of water, and you want to do this for three wells.  Shove samples back in freezer- unless you are planning on actually doing the next stage of the western today. In that case, shove them in a load of ice in an icebox and leave them on your lab bench.

23) Add 200 µl BCA assay to filled wells.

24) Add 210 µl BCA assay to three empty wells - these are your controls. Realise you didn't label them on the 96 well plate. Quickly do so with your lab marker.  

25) Take your plate to the hot plate storage thing that you still don't know the actual name off and incubate for half an hour at 37 °C. 

It looks like this. 

26) Go amuse yourself for half an hour. Might as well get a coffee and something chocolately to console yourself about having to do a western blot. 

27) Retrieve your plate and head to the spectrophotometer*

*Spectrophotometer is a piece of fancy equipment that measures how much light a substance absorbs or transmits at certain wavelengths. The more purple the solution, the more light it absorbs and the less light it transmits. We know how much protein each standard has and the spectrophotometer will give each of these standards with an absorbance value. We don't how much protein each of our samples will have - but we will get an absorbance values for each sample. We can compare these to the standard curve our standards give and work out our protein concentrations. 

Some labs just call it a plate reader. 

28) Try to remember how to work the flipping thing.

29) Remember what absorbance* you want. 

*Absorbance refers to the wavelength of light the spectrophotometer is going to measure. You can in theory do transmission, but absorbance works better. The purple complex that was created absorbs light 562 nm so using this wavelength will give you accurate data. 

30) Let your spectrophotometer do its thing.  Almost forget to save the file it generates with the relevant absorbance values. 

31) Discard the plate. After all that work you don't need it anymore. 

32) Attempt to use these absorbance values to generate a standard curve*

*Some spectrophotometers do this automatically. It's a curve that plots the absorbance values for your standards against the protein concentrations. 

Looks a bit like this, Credit to Odinity 2018.

33) Breathe a sigh of relief when you see the R² number*.

*This number represents the 'coefficient of determination'. It basically says how well the data fits, giving you an idea of how reliable it is. You want to be about 0.990 - 1.000. An R² of 1.000 is perfect. 

34) Forgot all you know about maths and try to calculate how much protein you have in your samples*

  *How it works is based on the equation Y = Mx + C which is what you use to work out the gradient of a curve.  Y is the absorbance, X is protein concentration, C is your y-intercept and M is your gradient. You can use the standard curve to calculate the M and C. Those values are what you then use to calculate the X of the samples. But then you also need to multiple by the dilution factor -or how much you diluted the samples by in the first place. So, if you put 10 µl

 of sample with 90 µl of buffer, your final volume is 100 µl. Divide 100 by 10 and you get a dilution factor of 10. 

35) With a long sigh of annoyance, start planning the next stage - the gel electrophoresis. FUN.

 

Honest Protocols! Making Lysates

 This is the second article in my new series; Honest Protocols, where I give a protocol for common lab techniques along with the realities of the protocol and the emotional changes they cause.

This week, I'm looking at how a lysate is made. 

A lysate is a liquid mixture that contains cell proteins, DNA and some other molecules usually contained within the cell- but it doesn't have the living cells itself.  This is done by breaking the cells open - called lysing and letting the molecules out. For most people, a lysate is often used for a western blot, but you can also use it for DNA extraction and protein purification. Lysates can be frozen so in theory you can forward-think and freeze some lysates in advance. Still, you have to make them at some point:

1. Pretend you are doing a simple splitting, and you aren't preparing for a western blot -one of the most annoying techniques that was ever invented. Decide to actually forward think for once - book a slot on the cell hood and remember to take your media, trypsin and PBS out of the fridge. 

2.Locate your cells in the incubator.  Look at them via a microscope, confirm there is enough of them and start preparing to split.

3.Carry out steps 10-16 from honest protocol 1 - Splitting cells. 

4. Find that this time, it’s not the flasks that have run out, it's the 15 ml tubes. 

5. Swear, run across to the storeroom, grab a bag of 15 ml tubes and run back to the cell culture hood.

6. You also find that the Eppendorf tubes* have run out and it never occurred to you to check them before you went to the storeroom. 

*Eppendorf tubes are tiny plastic tubes that are designed to have secure locks and seals to stop any contamination. You can get multiple types but the most common hold about 2mls of liquid. You can freeze them and centrifuge them. 'Eppendorf tubes' are a mouthful so you can just call them E-tubes. 

E-tubes. They also come in a load of colours. 

7.Stomp back to the storeroom, grab a bag of E-tubes and stomp back,

8.aYou can't work out how to open the bags neatly and efficiency, so just break it open with your thumb, grab what you need and spray it down with ethanol.

9. Repeat step 16-20.  Resuspend pellet with 1ml of PBS and transfer to an E-tube.

10.Prepare RIPA- protease inhibitors* mixture in a 100:1 ratio. Try to remember how to calculate ratios. Remember that you are going to need to chill the centrifuge* in advance so go and run it at 4 degrees for 10 minutes.

*RIPA buffer is what we use to get the protein out of the cell membrane and any tissues. It's a lysis buffer so it dissolves the cell membranes and releases the protein. But this process also causes natural enzymes called proteases to be released and these destroy the proteins. So, we add protease inhibitors to stop the proteases from acting. 

*The centrifuge being used here is different from the smaller one used in cell culture. Here, you are using a benchtop centrifuge which is much bigger. This gives it a much larger rpm, and you can also adjust the temperature. For this, you want the temperature to be low to stop the proteins from getting deformed. So we call it a refrigerated benchtop centrifuge. 

A refrigerated benchtop centrifuge. 

11. Look at the size of the pellet and attempt to judge how much of the RIPA-Protease you should add. You're supposed to add 'just enough' to resuspend the pellet. Eventually decide on 100 micro litres and call it a day. 

12. Vortex the E-tubes. Stick your finger in it when no one is looking.

A vortex. It's basically a mixer and

 makes sure the pellet is totally resuspended. 

Everyone sticks their (gloved) finger in it 

at some point.

13. Try to remember what rpm you're supposed to centrifuge at. Settle for 12,500 rpm for 10 minutes.  Brace yourself for the horror that is an unbalanced centrifuge.  Internally scream ‘KEEP THE LIQUID!' at yourself. 

14. Pipette liquids into fresh E-tubes. Realise that you really don't want to start your western blot today, so you decide to just freeze them for tomorrow. 

15.Discover that your cryobox* is full.

*Cryobox is just a fancy word for plastic boxes that store e-tubes and can resist high temperatures. 

                                            A cryobox. 

16. Work out what you can throw away in the ice storage box.

17. Stuff the e-tubes into the new spaces.

18.With the help of a coffee, return to your desk and start mentally preparing yourself for the fun that is a western blot. 

Honest protocols! Splitting Cells.

I'm starting a new theme on this blog: the honest protocols. 

These are accurate protocols on some of the things that scientists do on an almost daily basis - but with a twist. These honest protocols will describe the whirlwind of emotions that a scientist may experience along with the all the problems and delays that will inevitably happen even if the protocol sheet you were given doesn't tell you anything about them. I'm vaguely hoping it will be kind of educational for the budding scientists out there, maybe relatable for the experienced scientists and potentially insightful for those who are curious about what people might actually do in a laboratory. Hopefully, it will be a little funny!

First protocol up is Splitting cells. 

Splitting is what scientists have to go when their cells divide too much and need to be separated. Cells for experiments can be grown in these cell culture flasks that are designed to accommodate a certain number of cells based on the size. T75s can take about 8 million, and T25s can take about 3 million.  The cells are deposited in the flask along with some cell media that has all the nutrients they need, shoved in an incubator and left to it. The amount of cell media you can use is proportional to the size of the flask you use. So, if you let the cells divide for too long, you get too many in one flask and the media doesn't have enough nutrients to support them all.  This means that they will get stressed and start fighting. Sounds a bit like my old school's canteen at lunch hour. Cells being stressed can ruin a lot of data because they behave differently to unstressed cells. Explains most of my behaviour during my A levels to be honest. Anyway, if we want to keep our cells unstressed, we need to get them out of the flask and put some of them into a new flask with fresh media, where they can carry on dividing. Until we need to split them again. 

Cell culture flask sizes: The tiny one is T25 (surface area of 25cm^2), 

followed by a T75, a T175 and a T225. The lids often have a filter so the cells can get enough oxygen but stops anything that could contaminate the cells getting in. 

So step 1: Go to the incubator, locate your cells and check how they look under a microscope.  Cells usually stick to the inside of the flask and form a monolayer.  If you are going to split cells, you need to make sure you have enough of them to maintain a stable population- a small number of cells cannot always divide enough to give you enough cells for an experiment. But you also don't want to wait until the cells get stressed.  So, when looking at the cells, you want them to fill about 70% of your field of vision. 

This is about 70%. 'About'.

Step 2: Swear when you see that the cells haven't grown enough and you don't have enough for splitting.  You were going to use the left-over cells for a lysate and now because you can't split, you can't make the lysate- which you need for an experiment.

Step 3:  Experience the stages of grief and finally you accept that you can't split today. 

Step 4: Sulkily go to the freezer to see if maybe you froze some lysate you could use. You didn't.

Step 5: Stomp back to your desk to see if this delay is going to be completely mess up your life plan - or at least your plans for the next week. Grab a coffee on the way. Possibly a brownie if you need cheering up.

Step 6: Write up a new plan for the week and spend the rest of the day doing your data analysis. Who says you aren't flexible?

Step 7: It’s the next day! You slowly drag yourself up to the lab, bracing yourself for contamination and dead cells.  There's going to be a reason you can't do it today, you aren't going to be blessed with luck....

Step 8: Repeat step 1 - and the cells have grown! You're happy at first - and then sigh when you realise that you didn't book the cell culture hood in advance or warm up the reagents you will need. Cells can be fussy and don't like cold fluids poured on them. It can cause stress. Not sure I blame them.

Step 9: By some miracle, the cell culture hood booking sheet is empty*

*The cell culture hood is one of the most important pieces of equipment for anyone working with cells. It creates a sterile (clean) environment meaning you can open up your cell culture flask and know that unless you screw up, no viruses, bacteria or fungi can get into your cells and mess up your work. 

Standard cell culture hood. 

 You book yourself in for a slot that starts in about half an hour and start getting your reagents out of the fridge. You need Trypsin; an enzyme that is designed to get the cells of the walls of the flask.  Enzymes are made out of protein which get a bit deformed if too hot so that one can sit out at room temperature.  You need PBS - we use this to wash the cells from any proteins that interferes with the trypsin and keep the cells hydrated. You also need your cell media, which has all the components for healthy cell growth. You can add something called FBS which provides some extra proteins and some extra help - like taking multivitamins alongside a healthy diet. Antibiotics are also added to kill any bacteria.

Step 10: Wipe the entire hood down with blue roll* and ethanol. This is to kill anything that might be in the hood. The hoods do have UV light - which also kills everything and filter air, but you don't know if the person who used the hood before you actually used the hood properly. 

*What no one tells you before you step into the lab is that blue roll is actually the most important thing in a lab. Doesn't matter how many cells you have or what equipment you have, if you don't have blue roll, you are SCREWED. 

It's the exact same stuff that bars and kitchen use. 

Step 11: Remove the cell media from the cells.  For this you will be using a pipette buoy and a stripette. 

Pipette buoy on the left and a stripette. The stripette attaches to the buoy 

and the two buttons on the buoy control whether fluid is sucked up the stripette

 or down the stripette 

               
                                    

Step 12:  Wash the cells with PBS. If using a flask, you want anything from 5-10 ml depending on size of flask.  Question whether you sprayed your gloves down with ethanol before opening the flask.

Step 13: Pipette Trypsin onto the cells. 1-2 ml is usually fine. 

Step 14: Incubate for 3 minutes in the incubator.  Use this time to dead-scroll Facebook, mull over what you're having your lunch, question your life choices or make notes in your lab book. Realise that you didn't take your gloves off before touching your phone and you will have to put new gloves on before touching the flasks again. 

                                                        

Step 15. Check for a thick white fluid - this is the cells coming loose. Bang the flask on a table to shake them all loose- wince when you realise you should have 'tapped' and not 'banged'. Head back to the hood. Almost forget to spray the flask with ethanol. 

Detaching: When cells come loose. 

Can also tap the flask with your hand.

Step 16. Using your trusty pipette buoy, add your fresh cell media to the flask.  About 8mls. Transfer it all to a 15 ml tube. 

Step 17: Put 15 ml tube in centrifuge* Realise you need a second tube to balance it out. 

*Centrifuges are used to separate based on density. So, in this case, you want to get all the cells separated from the media and the trypsin. Centrifuges use gravity and centripetal force to pull the densest components down first, and because the cells are the heaviest part in this mixture, they will form a white pellet at the bottom of the tube. But because they use centripetal force, you need the centrifuge to be balanced either side. Which means we need another tube of the same weight. Shift though all the blanks* that some kind, thoughtful and organised lab technician made up.

*Blanks are a general term basically describing something that won't contribute to your results or give you data but will get your equipment working. In this case, the blank is a 15 ml tube containing some water. If we have 10ml of cell media + cells in a tube, we need a blank with 10ml of water. 

Step 18: After questioning your sanity for about 5 minutes, you find the blank. Shut your centrifuge, set it 1200 rpm at 4 minutes. Pray to whatever higher power you choose that it's balanced properly and today you won't hear the god-awful grating grinding sound of an unhappy centrifuge that makes you regret all of your choices. Use this time to mentally thank lab techs and return to dead scrolling.

*rpm means rotations per minute. Just tells you how fast the centrifuge is going. Honestly, the cells aren't that fussy, and the rpm doesn't actually matter that much. Just keep it below 1500 to stop them being damage.

Step 19: Remove the tube, check that you have a pellet. You do. If you haven't, something MAJORLY WRONG has happened. 

Step 20: Return tube to cell hood. Use pipette to remove the liquid - leaving the pellet behind. Accidently poke pellet with pipette edge. Tell yourself that its fine *.

*It 'probably' is.

Step 21: Curse when you realise that you didn't check whether the flasks you needed were actually in the flask storage drawer. They aren't.

Step 22: Run across to the storage room and grab a new bag. 

Step 23: Label your flask with your initials, the data, the type of cells and their passage number*.

*Passage number is how many times the cells have been split. If their passage number gets too high, they can get mutated and behave weirdly. 

Step 24: Pause as you try to remember what's the date and the passage number. Ask kind lab tech who walks in.  

Step 25: Consider if you have cool initials or whether your initials are just weird*

*I knew someone whose initials were EW. Her flasks were literally EW. 

Step 26: Pipette media into the new flask - about 9ml. Idly consider how when the lab tech showed you how to do this, it took them 5 minutes to do the whole thing and it has currently taken you about 15 minutes to even get to this point. 

Step 27: Return to pellet and resuspend it with 10 ml of media.

Step 28: Transfer 1 ml of this to the new flask, and add 9ml of fresh media*

*This means that you have one tenth of the cells growing in the old flask now growing in the new flask. 

Step 29: Make your lysate- with the remaining cells*.

*Honest protocol 2. 

Step 30: You decide to make up some media in advance, so hunt down your FBS from the fridge.

Step 31: You swear when you realise you used up your FBS last week.

Step 32: Retrieve the 50ml tube FBS from the freezer and mutter angrily when you realise that you need an hour to defrost it. 

Step 33: Scrawl your name on another slot on the booking sheet in an hour's time. Stuff the FBS in the water bath to defrost. Wipe everything down with ethanol.

Step 34: Come back to the lab after knocking back a coffee. Wonder if your entire blood stream is just pure coffee and sugar. 

Step 35: Wipe everything down with ethanol. Consider the sad fact that you have had to wait an hour to do something that will take you less than 5 minutes and it’s your poor planning and lack of forward thinking that led you to this. 

Step 36:  Pour the FBS into the media*

*You could actually be a bit more accurate if you feel like it. The media usually comes as 500ml and you need about 10% of FBS.  About. So, you could pipette 50ml of media out and replace it with the FBS or just shove in 50ml of FBS. 

Step 38: Return the cell media to the fridge. 

Step 39: Almost forget to put an extra FBS in the fridge- ALMOST. Grab the frozen FBS, shove it in the fridge.  You can use it next week when you need to make more media. For the tenth time this week, silently wonder how you manage to use up so much media. 

Step 40: Knock back another coffee... sigh and start planning the experiments you are going to do...