Hi there,
In my experiment I need to ligate 3 ssDNA, 80bp (S123) with help of 2 other ssDNA 80 bp as bridge (NS12).
this assembly uses single-stranded bridging oligos complementary to the ends of neighboring DNA parts, a ligase to join DNA backbones to assemble these 3 DNA fragments (S123).
Experiment process:
1) Ligation
2) PCR
3) Gel Electrophoresis
My concern is that hybridization bond between S123 and NS12 affects on PCR even with using no ligase.(attached figure)
In the attached figure you may see almost the amplifications are same between the samples does have ligase and the sample which doesn't have ligase.
The only difference is that at high concentration of DNA with no ligase some minor bands (100bp~120bp) can be seen (sample #8), while the minor bands hardly can be seen for the sample #7 which does have ligase.
I wounder if there would be a way to confirm the ligation or if there would be a way to denaturate NS12 and separate S123 prior to PCR.
Did you try to ligate two fragments first? check by PCR and then go ahead for the third.
To separate S123 from NS12, maybe you can try to work with biotinylated NS12 oligos and remove them after ligation through binding onto streptavidin beads and denaturation to release the S123 which can then be analyzed separately.
To confirm ligation within S123, one possibility could be to label S1 and follow its mobility shift by gel electrophoresis (*S1+S2+S3 vs *S1 or *S1+S2).
Hi Ali,
If I understand you correctly it may be easier to just do all of this in a single PCR reaction. The attached and the references therein may help you a little.
Good luck with your experiments
Andy
Hi Ali, I might be misunderstanding, but are there abasic sites that would result between the ligated oligos (according to your figure)? Perhaps PCR stalls on these? Maybe try DNA repair step after ligation?
Dears,
My concern is that hybridization bond between S123 and NS12 affects on PCR even with using no ligase.(attached figure)
In the attached figure you may see almost the amplifications are same between the samples does have ligase and the sample which doesn't have ligase.
The only difference is that at high concentration of DNA with no ligase some minor bands (100bp~120bp) can be seen (sample #8), while the minor bands hardly can be seen for the sample #7 which does have ligase.
I wounder if there would be a way to confirm the ligation or if there would be a way to denaturate NS12 and separate S123 prior to PCR.
Dear Philippe Frit,
Thank you for your attention, Biotinylation may a clue for my purpose i will try to do it. As I'm not familiar with this technique would you please send me a protocol?
if you just want to confirm ligation is there a restriction site in the double stranded dna that can be used to confirm annealing
have you tried to extract this band on gel and check by PCR? Or do a digestion after gel extraction if there's a restriction site in the double stranded DNA
Dear Arnaud Cheuk,
I did with extraction the band on gel and then PCR, the problem is that the hybridization bonding between S123 and NS12 may exist even after gel extraction.
According to the size I see on your gel, I think the assembly is done correctly with or without ligase after PCR. Sequencing with your forward and reverse primers could be an alternative to confirm your assembly.
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