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Trouble sequencing DNA from mutagenesis reaction - (May/04/2007 )

I have a large construct (~14 kb) that I managed to clone last year (9.4 kb plasmid + 4.2 kb insert!! Took a while) Somehow, there is some extraneous sequence right after the start codon in the vector (I wrote to the people we got the parent vector from, and they don't have information on the sequence of the whole vector, and said "Yeah, the sequence in the mcs is not exactly what you might expect." What? Helpful...) Unfortunately for me, this extra sequence contains a convenient stop codon right after the start codon!!

I tried Quikchange II XL to get rid of the extraneous 63 base pairs - nothing. Very few colonies, and any that grew and had enough DNA to sequence, had the extra bases!

I tried doing the mutagenesis in two steps, getting rid of half of the extra sequence each round. Nothing. Not with half, not with all. Nothing would grow.

I tried transforming and growing at room temp, just in case my insert is deleterious (it might be, shouldn't be expressing, but if there is leaky expression, it might make the bugs sick..) More colonies, but nothing sequences!

I tried mutagenizing out only the stop codon. I tried at RT and at 37. Again, some colonies, but nothing sequenced.

I have sequenced the parent vector no problem, with the same primer I am trying to use to check out this mutation. The primer is far outside the region I am mutagenizing. Even a primer for the end of the insert is not sequencing the putative mutants. I even did a maxiprep with a couple of the putative mutants, got a ton of DNA, it is the right size according to a gel, and NOTHING.

Help help help??? I am at the end of my rope, and am running out of ideas...

- Astrid

-Astrid-

this happened to me once on a recombination event. Can you PCR primers into it on each end with cut sites, do a quick one-step digestion and religation (be careful of framing!!) and abracadabra - site is gone!

Make sure restriction sites are not in gene.

Good Luck!

QUOTE (Astrid @ May 4 2007, 06:03 AM)
I have a large construct (~14 kb) that I managed to clone last year (9.4 kb plasmid + 4.2 kb insert!! Took a while) Somehow, there is some extraneous sequence right after the start codon in the vector (I wrote to the people we got the parent vector from, and they don't have information on the sequence of the whole vector, and said "Yeah, the sequence in the mcs is not exactly what you might expect." What? Helpful...) Unfortunately for me, this extra sequence contains a convenient stop codon right after the start codon!!

I tried Quikchange II XL to get rid of the extraneous 63 base pairs - nothing. Very few colonies, and any that grew and had enough DNA to sequence, had the extra bases!

I tried doing the mutagenesis in two steps, getting rid of half of the extra sequence each round. Nothing. Not with half, not with all. Nothing would grow.

I tried transforming and growing at room temp, just in case my insert is deleterious (it might be, shouldn't be expressing, but if there is leaky expression, it might make the bugs sick..) More colonies, but nothing sequences!

I tried mutagenizing out only the stop codon. I tried at RT and at 37. Again, some colonies, but nothing sequenced.

I have sequenced the parent vector no problem, with the same primer I am trying to use to check out this mutation. The primer is far outside the region I am mutagenizing. Even a primer for the end of the insert is not sequencing the putative mutants. I even did a maxiprep with a couple of the putative mutants, got a ton of DNA, it is the right size according to a gel, and NOTHING.

Help help help??? I am at the end of my rope, and am running out of ideas...

- Astrid

-biokmst-