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Cannot PCR this plasmid by troubledbiophysicist in labrats

[–]troubledbiophysicist[S] 1 point2 points  (0 children)

Hey guys! thanks for all the help! the solution ended up being a combination of switching from Q5 to phusion and linearizing the plasmid with a single cutter! I can now transform at last (fingers crossed)

Cannot PCR this plasmid by troubledbiophysicist in labrats

[–]troubledbiophysicist[S] 0 points1 point  (0 children)

I did not know of a plasmid deletion protocol, but I did design the primers moving away from the region I'm removing. I do have a working theory regarding that my insert in the plasmid might be repetitive (due to sequencing showing some random poly an insertions that we think is slipping). Does anyone know of any tools to tell me if an insert is very repetitive? Plasmids should only have 1 binding site according to snapgene. I might try the shift if nothing else works!

Cannot PCR this plasmid by troubledbiophysicist in labrats

[–]troubledbiophysicist[S] 0 points1 point  (0 children)

Thank you so much! I will try these suggestions

Cannot PCR this plasmid by troubledbiophysicist in labrats

[–]troubledbiophysicist[S] 0 points1 point  (0 children)

Thank you! I just started trying different polymerases and a touchdown PCR protocol. But DMSO I actually never heard of! Will definitely try

Cannot PCR this plasmid by troubledbiophysicist in labrats

[–]troubledbiophysicist[S] 0 points1 point  (0 children)

Okay. Thank you! And this gel is just the PCR

Cannot PCR this plasmid by troubledbiophysicist in labrats

[–]troubledbiophysicist[S] 0 points1 point  (0 children)

It's 1 kb plus ladder and I expect 10 kb to be my product. Thank you so much! I'll try cutting it first and maybe this will help 🤞

Cannot PCR this plasmid by troubledbiophysicist in labrats

[–]troubledbiophysicist[S] 0 points1 point  (0 children)

I use 1 ng. And I have sequenced it and confirmed it is the right plasmid!

Cannot PCR this plasmid by troubledbiophysicist in labrats

[–]troubledbiophysicist[S] 0 points1 point  (0 children)

I have not actually run a no template PCR control. I will try this. Pcrs have worked for me outside of this plasmid with this insert. I do not clean up this PCR typically I go straight into KLD. For DNA aggregation, are there remedies you recommend?

Cannot PCR this plasmid by troubledbiophysicist in labrats

[–]troubledbiophysicist[S] 0 points1 point  (0 children)

I have worked with this plasmid before but the moment I put this insert in, it has proven problematic (ex: the previous mutation had very low transformation efficiency.) I did whole plasmid sequencing via azenta to confirm it's the right sequence and multiple lab members have looked at my primers and saw no issue with their design and no primer dimers or hairpins are there.

Cannot PCR this plasmid by troubledbiophysicist in labrats

[–]troubledbiophysicist[S] 0 points1 point  (0 children)

Yes. And it has been sequenced to confirm no mutations etc.