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#1 2019-06-20 14:57:30

hyla
Member
Registered: 2019-06-20
Posts: 3

trouble with 2nd PCR! Please help

Hi,
I used the website to design the primers for 1st PCR. You can see the details here.
I sequenced the 1st PCR product and it seems that I have it correct however after the 2nd PCR (tried different annealing temp 61 to 68), I get colonies only with parental plasmid.
I would appreciate your tips and help.
the insert size is 672 bp long.
Best,
Hyla

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#2 2019-06-20 21:12:04

Steve Bond
Administrator
Registered: 2014-01-23
Posts: 124

Re: trouble with 2nd PCR! Please help

Hi Hyla,
It's difficult to trouble shoot with so little information. There are many tips and tricks discussed throughout the forum, so please see if there is any help there. If nothing seems similar to your issue, then post back with your full protocol and we can move on from there.
One thing that does jump out at me, however, is that you're trying to remove the better part of a KB of parental sequence. My experience has been that big deletions tend to reduce the efficiency of the reaction. Still possible, but harder.
-Steve

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#3 2019-06-23 07:24:17

hyla
Member
Registered: 2019-06-20
Posts: 3

Re: trouble with 2nd PCR! Please help

Hi,
thanks for your reply. the 2nd PCR reaction are as the following:
I then set up and ran the secondary PCR with the quantities of insert: plasmid and elongation time  indicated in the website using the 3-step protocol. I used Phusion polymerase.
The cycle was as follows:
98°C 30"
98°C 10"
61°C  30''
72 °C 3'
72 °C 5'
72°C 5'   
total of 35 cycles
would you suggest me to try 2 step PCR?
Thanks

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#4 2019-06-23 07:47:27

hyla
Member
Registered: 2019-06-20
Posts: 3

Re: trouble with 2nd PCR! Please help

Hi,
another thing: what did you mean by :"you're trying to remove the better part of a KB of parental sequence".
indeed the deletion /replacement that I try to do is about 700bp, is is too long? an and the insert is 672bp long.
it would be graet if you could comment on it.
Hyla

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#5 2019-06-23 12:41:59

Steve Bond
Administrator
Registered: 2014-01-23
Posts: 124

Re: trouble with 2nd PCR! Please help

Hey Hyla,
I would expect that you have caused a lot of high molecular weight artifact to build up in your reaction with this protocol. Try again, but reduce the extension step down to 140 sec and reduce the number of cycles to 15.
And I didn't mean anything more than what I said about the deletion. If you delete a couple dozen base pairs, it doesn't seem to have much of an affect on overall efficiency, but deleting hundreds or thousands of base pairs does. It's just something to keep in mind.
-Steve

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