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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
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
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
Hi,
I used rfcloning to insert four fragments including promoter to modified PBR322 vector.
At each step the insert was confirmed with colony PCR. Everything was fine.
Now I sequenced the plasmid and I could find only the parent vector.
I doubt the Dpn1 digestion step and I want to repeat the procedure.
I would like to ensure the primer designing is correct.
The project ids
ef07449f1b3d5033ea240b3527b4ffef
bc4d8dc5b02356491553fbb5db1545d4
fdf694539142f5f415e48286d0b4010c
Vidhya
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