I need to complain about life and science.
Yesterday, I did a DNA gel and behold, the BP reaction I did last week to transfer my attB-genes into a pDONR plasmid failed. I know this because the 3 different samples were digested with an enzyme that should cut one of them once, the second one 7 times, and the last one 3 times. I got 2 bands from all 3. I did the BP reaction to ensure the original attB-PCR worked, since when I ran a gel to test that, it looks questionable (but I wasn’t sure if that was because it failed or because the EcoRI and HindIII enzymes were left in a 4C fridge by undergraduates).
what does any of this even mean?
EcoRI (Eco-R-one) and HindIII (Hind-three) are restriction enzymes. This means they will find a segment of DNA (for EcoRI, it is GAA*TTC) and then cut at a particular point (so for EcoRI, it cuts between the second A and the first T, denoted by an * here). Depending on the sequence, a restriction enzyme may cut once, many times, or not at all. Traditionally, restriction enzymes are used to cut up DNA and then paste them together, but also can be used to cut DNA and run it on a gel so you get a nice clean band instead of a smear. I am using restriction enzymes to chop up DNA so I can identify it better on a DNA gel.
DNA gels are agarose gels, usually 0.5-2%, and are made with a special mold so there are wells. You put your DNA and a loading dye (mine is purple <3) into the well, and then an electrical current is run through the gel (and the bath it is in). Since DNA molecule are negatively charged, it will want to move towards the positive electrode in an electrophoresis box. Smaller pieces of DNA move faster than big ones, so the pieces of DNA are separated by size. They even make mixtures of known DNA fragments (DNA ladders) so you can see exactly how big your piece of DNA is by seeing where it migrates to on the gel compared to the fragments of ladder.
This refers to the BP reaction part of Gateway Cloning. You use PCR (haaa I am too lazy/tired to explain that right now, sorry) to attach an attB sequence to the end of your gene. Mix the product (so your attB-gene) with a pDONR plasmid and a special enzyme, let it incubate and pray to the science gods. The enzyme should use the attB ends to put your gene into the pDONR plasmid.
AND NOW I AM MOVING ON.
So my BP reaction failed, probably because the attB PCR failed. So today I set up to do gradient PCR for 2 of the 3 genes (this is because these 2 genes are likely to have a similar melting/annealing temperature, so the gradients should hopefully hit the best temperature for both). However, when setting up my negative control groups, I accidentally put the primer for gene A in with the template DNA of gene B. I still made a separate negative control, but jeez, I wasted so much template doing that. Also didn’t help that the lab tech was trying to talk to me about my life and I don’t want to share with her right now.
Next, I try to digest my original templates to see if I can at least get patterns of the genes alone. PvuII should cut gene A once, gene B 4 times and gene C 7 times. I digest, run a gel and it all looks good….except gene A cut twice. Or rather, there were 2 bands instead of 1. WHAT. WHY.
THEN the other grad student reminds me of some tissue samples that I started staining last week and they were just rotating in 4C for the last 5 days. ヽ(ー_ー )ノ Samples that are actually for the undergrad I am trying to help with research but they haven’t responded to my emails in the last week so….
Next, I go to grow some E.coli and make more of the template DNA I ran out of (because I used the wrong template) and I notice the only LB we have has something floating in it. I really should just throw it out, but I took some from the top. I will make more LB tomorrow and hope that my transformed E.coli and the antibiotic will kill any random thing in the overnight growth.
FINALLY the cherry on this crap-sundae is Mom texted and said she hasn’t eaten all day and she won’t make dinner for me and she is going to bed without eating. ಠ▃ಠ I told her to eat, and then she didn’t, so I went home and made her eat a cupcake for dinner. But I was looking forward to her cooking for me tonight…oh well.
I would really like to go shopping to ease all this stress. But I bought plane tickets to go to San Diego next spring for the Fly Meeting, and I won’t be reimburst for that until either the WC gives me money (I think I find out in December or January) or until I go to the finance department and have them take it out of my 2016-2017 grant money. I hope my boss doesn’t spend it all first.
And everytime I look back on my floordinate/bedinate posts, I am so annoyed with how wrinkled my bed is, and the dresses. Maybe this weekend I should try to iron everything.