cloning from PCR - (Jun/24/2004 )
what seems strange is after digesting, the vector (~3.2kb) shows a clear exised band of about 40bp, the fragment between BglII and XbaI, whereas my digested PCR product shows no small band, even when I ran a 20% acrylamide gel to look for extra small fragments (5bp and 8bp from each primers' 5' end). is this something i should be worried about? how can the enzyme sites NOT be there when I get a very dense band from my PCR reaction at 1.1kb?
man, how can you see the bank about 40 bp, 4%argrose gel?, i am going to check my double digested dna between BamH1 and xho1 (40bp) now, so need your suggestion, thank you.
Hi,
The RE Xba I is sensitive to dam methylation. Maybe a reason why you don't see any small fragment is because one of you RE didn't cut...
I believe there is some problem with your transformation procedure. Include a positive control to check the technique.
You are uisng 10ul of ligation mix with 100ul cells. that is 1:10 or 10%. That I beleive is way too high. try with 1ul and 5ul.
From my experience 6 bases on the end is definately not enough with regards to digesting ends of PCR product for anything except maybe EcoR1. You could try blunting it in to your vector, or if you don't want the extra few bases blunt into a cloning vector and cut out of there to insert into your vector.
Cheers,
TA-cloning would be also my advice. Have done the same thing, created HindIII and BamHI sites, but couldn't cut ligate it with the vector. I have cloned it into pGEM-T-easy and cut it out, then the ligation was no problem, many clones!
I would recommend extending the ligation time and increasing the ligase amount.
The RE Xba I is sensitive to dam methylation. Maybe a reason why you don't see any small fragment is because one of you RE didn't cut...
xba1 digestion overnight works wonder !!! try this ..
what seems strange is after digesting, the vector (~3.2kb) shows a clear exised band of about 40bp, the fragment between BglII and XbaI, whereas my digested PCR product shows no small band, even when I ran a 20% acrylamide gel to look for extra small fragments (5bp and 8bp from each primers' 5' end). is this something i should be worried about? how can the enzyme sites NOT be there when I get a very dense band from my PCR reaction at 1.1kb?
I don't know about visualizing the small pieces, but you can perform the digestion right in the PCR reaction mix. Both enzmes are compatible with primer extension mixes. This may not help, but we have experienced some plasmid/insert combinations that are incompatible. We have sequenced both pieces and ligation/transformation still does not work. What plasmid are you using? Is it an expression plasmid?
what seems strange is after digesting, the vector (~3.2kb) shows a clear exised band of about 40bp, the fragment between BglII and XbaI, whereas my digested PCR product shows no small band, even when I ran a 20% acrylamide gel to look for extra small fragments (5bp and 8bp from each primers' 5' end). is this something i should be worried about? how can the enzyme sites NOT be there when I get a very dense band from my PCR reaction at 1.1kb?
When transforming, your ligation should not be more than 5%. If you're using 10uL ligation, use 200uL cells. Or, my suggestion, use 0.5uL ligation and 10uL cells. Ligate for 2 hours.
im designing almost same experiment and im setting 3 bases extra for EcoRI and 3 bases for XhoI for the primers...that is more than necessary according to New England Biolabs and also other sites.....am I wrong?
