simple pcr questions - simple pcr questions (Oct/03/2002 )
Hi. Need help on some really basic pcr steps.
Once I have my 50ul reaction micture, how much of that do i put in my actual pcr reaction tubes (the ones that go in the cycler)?
Also what is difference between DEPC and deionized water?
Also, once i have my pcr cycler set up and running, when do I start making the agarose gel? Can i start right away and let it solidify or is it not good to make it too early and let it sit?
Finally, what is a general note or rule for working with chemicals, should i always try to keep everything in the ice bucket?
You can put all of it in a reaction tube (some use 25ul, other 100ul). I always use 50ul.
DEPC=di-ethylpyrocarbonate (destroys RNases) --> used when working with RNA to prevent degradation
You even can make the gel the day before as long as you put it in the running buffer so that it doesn't dry out
Yes, especially the polymerase. You best divide the NTPs in different aliquots so that you don't have to defreeze and freeze the hole amount each time