50ml of 1xTAE buffer with 5ul of Sybr Safe (10000x in DMSO) and 0.4g of high quality agarose
microwaved for 2 min
poured into electrophoresis apparatus with 10 well comb (from Owl)
running at 150V for ~40 min
So the PCR reaction didn’t work again. Hmmm. My first measure of adjustment is usually to adjust the annealing temperature so I’ll start there. Troubleshooting PCR is so annoying, but I want to get some product before next week so work at it I will.
Yesterday I setup a PCR reaction using both our labs thermal cycler from Thermo and our OpenPCR thermal cycler. I want to make pALS PCR fragments (4kb in length). And here are the results of that experiment (with setup):
50ml 1x TAE buffer mixed with 0.4g High Resolution DNA agarose
heated in microwave for 2 min
added ethidium bromide (EtBr) to final concentration of 1ug/ml
gel cooled for 40 min in fridge
Meanwhile, 5ul of 9 of 11 PCR reactions put in PCR tubes with 1ul of 6x loading dye added (for 6ul total per tube)
the tubes selected were 4 tubes from OpenPCR (numbered 1-5) and 5 (of 6) tubes from Thermo thermal cycler (numbered 6-11), so tubes 1-4 and 6-10 were selected for gel analysis. This is because there are only 10 wells and 1 well is needed for DNA ladder.
Once gel cooled, 250ml of 1x TAE added to electrophoresis device (what’s the name of this thing?), filled to cover gel
6ul of each of the 9 prepared tubes were placed in the wells along with 6ul of pre-prepared 1kb DNA ladder.
connected to power supply and run at 150V for 45min
Based on the images above, the PCR reaction was a failure. Lanes 2 and 4 have very feint bands where the 4kb product should be, indicating the reaction succeeded but not to the degree required. This could be for any number of reasons, but most likely due to inactive enzyme, old reaction buffer, degraded dNTP’s, etc. Basically I’ll be doing this reaction again next week when all my new supplies come in.
Notes about the image acquisition:
Since I ran the gel with the EtBr added to the molten gel, I did not need to run the gel and then add EtBr. I also did not destain the gel after completion of electrophoresis. The images above were captured with my phone camera. To see gel results typically I use SybrSafe from Invitrogen with their special illuminator, but since I used ethidium today I had to use the hand held UV lamp we have. This means worse quality photo, but oh well.