...and the second primer worked the first time I used it as well. With the correct temperature too. Wow! I am feeling content and happy now, yet I still believe something is wrong. Or at least it will not work all the way... ah well, I'll skip my [realistic] pessimistic view of it all and try and purify the pcr products tonight so I can run the third [VERY] important pcr tomorrow morning. Actually, I am pondering staying in lab for another four hours and do the pcr tonight but nahh... I should sleep some and then of course it's the dreaded 'being tired and sloppy' which I really don't want to be today! Not when it is working!! (small dance of joy!!)
off to clean the products.
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update> I stayed (not really surprised) and put a reaction over night. I did, however, elute the purified products into 50 microliters .... I should have remembered that concentration is the key. Tomorrow I'll redo it all and remember to elute into 25 microl maybe? hm, I'll think about it. All of it probably since I didn't gel purify but rather did a quick pcr prep purification. I wonder if that is a big difference, really? Anyone knows?
2 comments:
Well, you are probably long past those PCRs, but you can often speedvac your elution products and use them (I don't know what your final destination is and it depends).
It's so gratifying when successes of any size start to chain up, one after the other - easy to get drunk on it. I totally sympathize with wanting the results of that third or fourth PCR right NOW. I remember being appalled at the first postdoc who worked with me, who stayed until the end of his PCR and put it away with a poured gel in the fridge, to migrate in the morning. How could he not want to spend 20 more minutes and know the results?! End result, he is earning twice as much as me at UCB Pharma. But I bet I'm just as happy if not more so ;-)
hehe, I know what you are saying.
Well, the pcrs seem to be ok, it ismore a factor of keeping good concentrations after purification and mixing into the longer fragment but I think I have got it ;)
Just need to do some more reactions at the same time, say three of the same in order to pool them and then ligate.
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