small sized restricted bands couldnt be visualized
Posted 10 March 2005 - 07:57 AM
I digest for around two hours, but I have digested for up to 16. If I'm too busy to run the gel I freeze but usually I run them right away.
Posted 11 March 2005 - 12:13 AM
i well try to put th restriction enzyme with different concentrations starting from ,5 ul till 1ul and incubate for only 2 hours in 37 C incubator after adding the enzyme
by the way whats ur annealing temp Jim?? and do u have photos of what bands to see in the hetero and homozygotes after restriction ?
thanks Fred and Jim
Posted 11 March 2005 - 06:19 AM
My rxn temps are:
I have photos; how do we go about sending pictures within this forum?
Posted 11 March 2005 - 07:25 AM
thanks for replying
i m new to this forum so i dont know how to send photos at it ...but if it would help u can send the photos as attatchments on my email
if its not so much trouble
Edited by lula, 11 March 2005 - 07:25 AM.
Posted 11 March 2005 - 08:49 AM
Hope all is going well,
Posted 11 March 2005 - 08:58 AM
thanks alot JIM
i performed a testing PCR yeaterday but my annealing is 51
tomorrow i well incubate with Mbo 11 for 2 hours and test on agarose 45 to see what well appear
thanks for ur help
i well tell u all what happens
Edited by lula, 11 March 2005 - 09:01 AM.
Posted 14 March 2005 - 01:00 PM
How did your MTHFR run go? Anealing at 51 C might be ok; I've seen protocols that call for 55C. I Run Factor V Leiden, Prothrombin II, and MTHFR on the same thermocycler program and 65 fits them all pretty well.
Posted 14 March 2005 - 01:58 PM
im thinking about raising the annealing temperature from 51 to 57 because i have been getting a smear in the PCR BAND lately
(my primers Tm is 64 )
as regard the restriction ,,,still no luck
but i didnt give up yet