RF-Cloning.org Forum

vithya

Member

Registered: 2014-10-23

Posts: 4

suggestions for primary PCR

Hi
I am currently trying to insert a 63 bp linker to the modified PBR 322.
I am not able to get a clear product in primary PCR.
Initially I used 25 pmoles of each primer with 25 pmoles of template (63 bp longmer). the band was diffuse with dimer.
I reduced the primer concentration to 2.5 pmoles and template to 10 ng . still the band is very faint and I am unable to purify it for secondary PCR.
I am using 5 cycles for primary PCR.
Can I increase the no of cycles to get a clear product?
what would be the optimum template primer concentration?
This is my project id  917bbeb588f2dea62ce7ea31531c1e08
I would like to know any problems with the primary.

With regards
M.Vidhya

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: suggestions for primary PCR

Hi Vidhya,
While your insert is relatively small it is not completely synthesized by the primers, therefore you should run a normal 30-40 cycle primary PCR reaction. Aim for a final concentration of ~0.2 μM of each primer and drop your template to ~0.1 ng.
Hopefully this solves your problem.
Best of luck,
-Steve

vithya

Member

Registered: 2014-10-23

Posts: 4

Re: suggestions for primary PCR

HI steve,
Thanks, the primary PCR worked well with 35 cycles and I got a clear band.
But I did’nt get ant colonies in secondary PCR.
Secondary PCR  reaction was set up with
5X Phusion HF       -  4 µl 
10 mM dNTPs      -  0.4 µl
Megaprimer         - 0.7 µl (25 ng)
Plasmid             - 2.5 µl (45 ng)
Phusion
DNA  Polymerase -  0.2 µl
NFW to 20 ul
ThePCR conditions were
98 – 30 sec
98 –   8 sec
62 -   15 sec
72  - 1.15 sec  15 cycles
72 – 10min

Dpn digestion was done with 20 units for 2 hours at 37◦C, then I ul of the reaction mix was transformed in BL-21 cells and plated in tetracycline plates.
I would be happy to know where I am going wrong and how can I improve the secondary PCR.
M.Vidhya

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: suggestions for primary PCR

Hmmm... On your next try, keep the conditions pretty much the same, but try increasing the extension time to 1:45min.
Are your comp cells home grown? What is their transformation efficiency with control plasmid (a nice control is to take out a microliter of PCR mix before DpnI digestion, and transform with that)?

vithya

Member

Registered: 2014-10-23

Posts: 4

Re: suggestions for primary PCR

Hi Steve,
I am back after a long time with some results of this project  which I would like to share.
With some improvements in Primary and secondary PCR and with fresh comp cells I was able to get positive colonies (confirmed by colony PCR).
Now  the sequence results showed 6 gaps and I mismatch in the insert that was cloned.
The insert is a 63 bp polylinker with restriction sites.

Observed sequence   ---GCGCGCCTTCCCCCGGCAGG------ CCTCGACGCGTCACA-----GTTCAGCTAAGCCGCCTTAAG
Expected sequence    ---GCGCGCCTCCCCGCGGCAGGGGCCCTCGACGCGTCACACCGGTTCAGCTAAGCAGCCTTAAG

Are the gaps due to secondary structures with in megaprimer because of GC rich insert.
Can this be checked while primer designing.
Any change in PCR conditions will help?
Any suggestions to overcome this.

With regards
Vidhya

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: suggestions for primary PCR

Hey Vidhya,
I'm glad to hear that you've made some headway
It's really strange to see this pattern of deletions from your insert sequence. Did you send multiple clones off for sequencing, and all of them came back the same? If that's the case, then the original insert sequence you amplified during the 1° PCR might actually be the culprit. If you're pulling the poly-linker out of another plasmid, I'd suggest sending that off for sequencing.
If, on the other hand, you've already considered this... I'm really not sure what's going on. Secondary structure could certainly interfere with the PCR reaction efficiency, but I wouldn't expect it to cause a predictable pair of deletions.
Is there any other info you can think of that might be relevant?
-Steve