When I digest the ends of the segment & the plasmid with restriction enzymes, they can not ligate together and although some times I have clones when I use colony PCR I do not have any band.
I probably could help if I had the complete info... Plasmid, insert, enzymes used and your whole protocol.
Benjamin! thank you for your attention.My plasmid is pRNAi vector,my insert is 80bp siRNA, and BamHI & HindIII are my two enzymes.
my fragment is in T-vector.I cut it with BamHI & HindIII . Also I cut my pRNAi with the same enzymes. but when I ligate them and transform to the bacteria, and use colony PCR with specific primers ,present colonies do not have the band of my fragment.I think because the plasmid is too big & the fragment is very small , they can not find each other!!.
JOrg is right...u will have to increase the amount of pRNAi that u digest. Also we faced problems when we used oligos with overhangs. For us the blunt ended oligos worked better..
I first pcr the pRNAi & fragment-plasmid . then I digest them with RE , ligate & transform to the bacteria.So, I think I do not have the problem of the amount.Also I can not use another enzymes becuse I use pRNAi vector that has just two enzymes (Bam HI & Hind III) in its MCS for cloning.
From Jörg:
Maybe you purification method of the DNA-fragments does not work well or the colony-PCR has a problem. You can order oligos for the 80 bp fragment in the way that they have the BamHI and Hind III overhangs after anealing them.
I think that would be a very good idea, I used to clone all my shRNA cassettes (pSuper) with that method... If you still want to subclone: be sure to use the right method (e.g. a column that can purify your 80bp fragment) and make sure to run a gel after to see your digested Vec and ins. Nanodrop the DNA (better Qbit, picogreen...). It's essential to know the integrity and concentration of your fragments. Use 50ng Vector with 0, 1, 5, 10 and 20 ng of insert (to try different ratios) and ligate them with a quick ligase (PEG enhanced ligation) for 30min @ RT. Followed by transformation (long procedure, 5mul ligation reaction to 100mul Bacteria, STBL3?). Fuge down cells and plate them all! Voilà...
Be sure to do a digestion after minis and run at 3% agarose. PCR might not work through the hairpin, especially with high GC content in the stem.
Good luck!
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