It could be as John suggests, that it is a sequencing problem, which seems most likely for SNPs/ mutations in the vector, or damge to the vector by UV exposure as you suggest.

Before repeating doing anything else, you really need to know if the SNPs/ mutations are genuine.

1)Could these be present in the original plasmid vector - do you have sequencing data from your own lab, or are you relying on sequence data from the manufacturer? I've used plasmids from some manufacturers (no names mentioned) that have several undeclared restrictions sites present, as well as multiple sequence inaccuracies.

2)How long is the good quality sequence read? Check the chromatogram, not just the sequence printout. If it starts looking wobbly of has obvious miscalls or nucleotides that cannot really be called before the SNPs, then the region in which the SNPS are needs to be discounted.

3)If you really want to know what the sequence is then you have options:

-if the sequence is relatively short ( under 6-700 bp and not excessively gc rich or containing hairpins) then you may want to repeat the sequencing with more/ better quality DNA.

-as John suggested, try to sequence using a primer on the other side of the SNPs from the original sequencing primer, or using a primer further forward in your insert, but ideally with area you want to examine at least 100bp away from the primer as the sequence can also be inaccurate in the first 100 bp as well as at the end.

-also if you have a plasmid that has hairpins or supercoils in such a way as to make sequencing of your area difficult, then sometimes cutting with a restriction enzyme that cuts away from your primers and area of interest can make for better sequencing.

Finally, if you confirm that your clone does have multiple mutations in your vector but not your insert then at least you can cut out the insert and reuse, but with short uv exposures.

If you want to avoid uv exposing the vector, then you could use PEG8000 /MgCl2 precipitation to remove short DNA fragments from cutting the multicloning site (https://openwetware.org/wiki/Size_selective_DNA_precipitation). This can also help remove digested/degraded RNA from small scale plasmid preps without columns. It would potentially also work in reverse to keep the insert but not the vector in the supernatant and avoid the need for a gel prep of your digested insert (if small), although I havent tried this myself.

{edit - I found that using this protocol to eliminate RNA worked well on small scale preps, but was inefficient and slow when used on large volume alkaline lysis preps from 500ml-2l flasks) as it took days at 4C for DNA to stop precipitating and seemed to require higher proportions of the PEG/MgCl2 solution to precipitate more DNA}

If you are fortunate and have a restriction present in the portion of multicloning site removed from the empty vector, but not in the vector with your insert, then you could add 1ul of the restriction enzyme 5-10 minutes before transformation to reduce the numbers of empty vector coming through.

Sorry for the rambling and hope some of this is helpful.

Hello, if no any problem with procedures that you mentioned annealing temperature etc., may be some DNAase in your reaction mixtures cause problem.

Karen A. Darbinyan could it be that my vector was a little overexposed to UV light?

Could it simply be poor sequencing? Sequencing quality declines the further you get from your primer. Do you see the SNPs/insertions when you sequence from both directions?

Hello, depends from wavelength UV can cause real damage of DNA.

John Hildyard hmm the little SNPs are found rather close to the start of my plasmid though. My mentor said it could be that my vector was exposed to too much UV light when I was performing gel extraction.

Are these mutations consistent i.e present in every clone?

Have you tried testing controls:

1. Digested plasmid, then re-ligated and transformed

2. Undigested plasmid

Carry out at the same time as other ligations. Sequence. Do you get the same mutations. What I'm trying to see here is if the mutations are driven by the presence of the insert.

Try a different strain, XL1Blue/DH5a

Ian R Wilkinson They do seem pretty consistent. I haven't tried testing controls. Maybe I'll do that. Thanks!

For DNA isolation we run a small sample on the inside lane (Main gel), then the remainder in the peripheral lane: This peripheral lane is cut from the gel prior to exposure to UV. A photo is taken of the main gel and the desired DNA is excised. Line up the peripheral lane with the main gel and cut out the desired DNA band by reference to the main gel. This way you do not over expose DNA to UV. The other way is to use a safe stain like Midori green and cut the band out under safe light.

It could be as John suggests, that it is a sequencing problem, which seems most likely for SNPs/ mutations in the vector, or damge to the vector by UV exposure as you suggest.

Before repeating doing anything else, you really need to know if the SNPs/ mutations are genuine.

1)Could these be present in the original plasmid vector - do you have sequencing data from your own lab, or are you relying on sequence data from the manufacturer? I've used plasmids from some manufacturers (no names mentioned) that have several undeclared restrictions sites present, as well as multiple sequence inaccuracies.

2)How long is the good quality sequence read? Check the chromatogram, not just the sequence printout. If it starts looking wobbly of has obvious miscalls or nucleotides that cannot really be called before the SNPs, then the region in which the SNPS are needs to be discounted.

3)If you really want to know what the sequence is then you have options:

-if the sequence is relatively short ( under 6-700 bp and not excessively gc rich or containing hairpins) then you may want to repeat the sequencing with more/ better quality DNA.

-as John suggested, try to sequence using a primer on the other side of the SNPs from the original sequencing primer, or using a primer further forward in your insert, but ideally with area you want to examine at least 100bp away from the primer as the sequence can also be inaccurate in the first 100 bp as well as at the end.

-also if you have a plasmid that has hairpins or supercoils in such a way as to make sequencing of your area difficult, then sometimes cutting with a restriction enzyme that cuts away from your primers and area of interest can make for better sequencing.

Finally, if you confirm that your clone does have multiple mutations in your vector but not your insert then at least you can cut out the insert and reuse, but with short uv exposures.

If you want to avoid uv exposing the vector, then you could use PEG8000 /MgCl2 precipitation to remove short DNA fragments from cutting the multicloning site (https://openwetware.org/wiki/Size_selective_DNA_precipitation). This can also help remove digested/degraded RNA from small scale plasmid preps without columns. It would potentially also work in reverse to keep the insert but not the vector in the supernatant and avoid the need for a gel prep of your digested insert (if small), although I havent tried this myself.

{edit - I found that using this protocol to eliminate RNA worked well on small scale preps, but was inefficient and slow when used on large volume alkaline lysis preps from 500ml-2l flasks) as it took days at 4C for DNA to stop precipitating and seemed to require higher proportions of the PEG/MgCl2 solution to precipitate more DNA}

If you are fortunate and have a restriction present in the portion of multicloning site removed from the empty vector, but not in the vector with your insert, then you could add 1ul of the restriction enzyme 5-10 minutes before transformation to reduce the numbers of empty vector coming through.

Sorry for the rambling and hope some of this is helpful.

Dear Rachel,

You can use Pfu DNA polymerase to amplify your PCR product, it can avoid error during PCR amplification before you insert into vector. .

Thank you, everyone, for your inputs. I noticed this other problem when I check my sequencing results as well. Every time I send my plasmid for sequencing, the sequencing company keeps telling me that there are overlapping peaks and the sequencing reads have been very bad. Previously, I'd sent the same vector backbone with different inserts and the same primer as well, the reads were great. What do you think could be the likely cause?