doing PCR using PCR product , help plz !!
Posted 04 February 2013 - 06:03 AM
let me get straight to the point . i am running out of my template DNA so i decided to use the pcr product of it as a template for another pcr .
so i used my main template DNA for the pcr . after my pcr was done i used 5 microliter of it and added it to a new pcr kit , then i added 15 microliter distilled water , and 1 micoliter of each primer . and ran the same program that had given a very good pcr product in the first place .... but , it didnt work !!! and i a m really worried plz guys if u know any thing that could help me , do tell !
Posted 04 February 2013 - 06:19 AM
I usually add 1 in 100 dilution or even 1 in 1000 dilution from the first PCR product to do the second PCR.
If you start from too much DNA from your 1st PCR, all the primer you've added might have used up from the first cycle therefore no PCR reaction will occur.
I hope this will help
Posted 04 February 2013 - 10:29 AM
wish me luck
Posted 06 February 2013 - 04:54 PM
2) I would also concern about the error rate in your amplicons. I don't think the quality will be good if you intend to do DNA sequencing later.
Just my 2 cents.
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"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
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Posted 08 February 2013 - 05:46 AM
Then for the 2nd PCR, I just take 1 uL of the PCR product and run for another 25 cycles. It usually works very well!
But keep in mind that your first PCR has to have a good quality (no unspecific bands). And if you want to sequence this fragments, always use a proofreading polymerase, Nested PCR will always increase the chances of mutations.