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small sized restricted bands couldnt be visualized


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22 replies to this topic

#16 pcrpetie

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Posted 10 March 2005 - 07:57 AM

After I run the pcr rxn, I add 0.5 ml of the restriction enzyme (5000U/ml) to each rxn tube. So the only buffer comes from whatever NEB puts in the tube with the RE. I don't know how, or why, this technique was initiated; my predicesor was not around when I came on board. It worked, so I kept it.
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.

#17 lula

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Posted 11 March 2005 - 12:13 AM

HELLO


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

#18 pcrpetie

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Posted 11 March 2005 - 06:19 AM

Lula,
My rxn temps are:
95 C-2min
(94C-30";65C-30";72C-30")X35
72C-4min
I have photos; how do we go about sending pictures within this forum?
Jim

#19 lula

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Posted 11 March 2005 - 07:25 AM

hi Jim


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
dr_genome1@hotmail.com
if its not so much trouble
thanks again

Edited by lula, 11 March 2005 - 07:25 AM.


#20 pcrpetie

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Posted 11 March 2005 - 08:49 AM

No problem, I'll send them from my home computer; I don't have a scanner here and the're polaroids.
Hope all is going well,
Jim

#21 lula

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Posted 11 March 2005 - 08:58 AM

hello

thanks alot JIM
i performed a testing PCR yeaterday but my annealing is 51 :ph34r:
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.


#22 pcrpetie

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Posted 14 March 2005 - 01:00 PM

Lula,
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.
Jim

#23 lula

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Posted 14 March 2005 - 01:58 PM

hello

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 B)




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