5 replies to this topic

[M_shabani_medbiotech]

hi every one
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 !
regards
Mina

[Lisa Hsu]

I think you might have added too much DNA for your second PCR.

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

[pcrman]

I agree with Lisa Hsu, you need to dilute your fist pcr pruduct before using it as template for the 2nd PCR reaction.

[M_shabani_medbiotech]

thank u so much guys , tomorrow  i'm gonna do as u said . and i'll keep u posted
wish me luck
xoxo
Mina

[Adrian K]

1) Another possibility of the reaction failure is due to too much carryover salt.
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.

[vilperte]

I usually start with 50ng in my 1st PCR, and it runs for 25 cycles.

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.