I am trying to clone my PCR products. I perform double digest (XbaI, NotI) for my plasmid vector (5.3 kb) (Our stock pET28b (+) exist in DH5alpha)and insert gene (1.0 kb). I perform ligation using T4 DNA ligase, incubate for overnight (16-18) at 13 C, and then transform in TOP10. I've been using 3:1, 5:1, 10:1 or more insert: vector molar ratio.
I do this many times and checked all enzymes and buffers and changed them. I am sure about what I did with everything before transformation.
After picking colonies I followed Qiagen protocol for extraction with miniprep kit.Then I checked plasmid DNA transformants with digestion. But I get pDNA transformants with size smaller (4 kb)than my original pDNA vector.
Can anyone help me?
thanks
The digestions with these two enzymes were did separately with the right quantities per DNA? And because the enzyme specificity can bring problems, for the incubation (overnight) try to use the minimum time as possible stipulated by the manufacturer.
I did double digestion for two enzymes above because their buffers was same(buffer H Roche) for two hours.
Morever I used two different enzymes(BamHI and SacI in seperately reaction for this test) too. But I get same result after picking colonies by other hand again I had smears and 4 kb pDNA transformants. Surprisingly I couldn't cut pDNA transformants with BamHI and SacI this time but NheI cutted( 4 kb). PCR was positive for insert gene( additionaly I have to mention I didn' t PCR for other pDNA transformants).
Hi there,
Why don't you use PCR on clone as screening with T7pro and T7ter primer set?
Using XbaI and NotI with pET28 results in the deletion of RBS and initiation codon of the plasmid sequence, so you have to bring them within your fragment to clone...
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