I am trying to clone a 200 bp fragment (blunt ended) in pBluescript SK vector (Sma I digested) using NEB T4 DNA ligase. But not yet successful with it. Please suggest.
Can you explain more. Describe each step and what you have observed so far.
Do you really have to clone with blunt end single digest?
It's much easier to clone with two incompatible sticky ends since there will be less re-ligating of vector and your insert will be in the right direction.
I have PCR amplified the 200 bp DNA fragment (BLUNT ended) and gel purified it. The pBSK vector is digested with SmaI and gel purified. Have been trying to ligate the 200 bp insert in pBSK. But getting no colonies. The positive control (pBSK undigested) is giving colonies. To increase transformation efficiency I am also doing electroporation of E.coli. But still no colonies when transformed with the ligation mix.
Have you checked that you are not using a polymerase that adds an A overhang to the 3'end, such as Taq? Have you phosphatase treated your vector, since I'm surprised that it didn't re-ligate after ligase treatment, in which case you will need to add phosphate group to your PCR product. Are you using high transformation efficiency bacteria? I use 10^9 cfu/ug for tricky cloning.
That is what is bothering me the most that I am not even getting religated vector colonies.
Fresh ligase? Add fresh ATP to buffer. It usually goes off quickly
As Amanda has suggested, you may need to kinase (T4) your PCR product. It can be added to some ligation mixes if you check the buffers
ATP is pretty labile. You could try adding PEG (it is present in the Promega rapid ligation kits for example). Are you doing ligation at r/t , 4C and for how long? For blunt end cloning, I have used CloneJet pJet vector which is quick (5-20 min), does not require a PCR clean-up (1ul PCR required) and does not need a colour selection (has a poison sequence - only inserts survive). If you think there is a problem with the ligation, try a PCR with the M13 /T3/T7 sequencing primers to verify ligation. You should only have to do 10-20 cycles.
Thanks Amanda and Ian for your suggestions. I am using Phusion enzyme for blunt ended PCR product. Even I have tried fresh ligase and buffer. I am ligating at 16 C O/N. Also tried 2 hr 25 C followed by 16 C O/N. Can you suggest some temperature variations for ligation which I may try.
I'll try your suggestions too.
Your ligation conditions do not seem unreasonable though I have used 4C o/n - I am generally in favour of PEG mediated ligations as these are faster.
For reasons that I have never really understood the rate of success with gel purified fragments in ligations seems to be lower than using unpurified material. Whether this is due to some residual nuclease activity in gels or as a consequence of the subsequent purification steps and carry over of inhibitors, I don't know. As a last resort (not using gel purified material, - just PEG or EtOH ppted) you could cut and phosphatase your vector, kinase your PCR product (assuming that it is normally a single band), mix aliquots of the 2 (say 10ul ea) and PEG ppt them. PEG pptn is quite effective at getting rid of small nucleotide sequences. You should end up with a pellet of concentrated co-ppted vector and insert which is redissolved in a small volume and used in 10 ul ligations followed by transformation
You may have to accept a higher level of religated vector but it may be a step on from not getting anything.
Thank you so much Ian. I'll try PEG pptn and ligation as you have suggested.
Hi Amanda and Ian...finally the ligation worked ..got few positive colonies. I tried multiple vector, insert ratios and 1:7 at 16 C ON worked.
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