I"ve been doing a cloning about 4 months already. i've done all the controls and I 've used differents vectors and also I've done vector dephosphorylation though I used double restrictions enzymes. i've used differents kinds of primer believing that maybe the base between the end and recognition site were not much , so I've assured that the bases at the end can at least be 7.the pcr always gives a very good band of the fragment and I've tested differents time of digestion but still I don't get the ligated fragment. the vector doesn't have problem because we tested with others fragments and it works. where can be the problem of my fragment?
Maybe fragment is toxic? therefore you cannot get proper ligation. Maybe add a little bit more info about what you are trying to clone and into what type of vector. Also add what enzymes are you using and what strain you using for transformation.
If your fragment expresses a toxic polypeptide, you should use a strain that affords tight control of expression, e.g. BL21AI (arabinose-inducible). Otherwise, how did you purify your PCR fragment before performing the digestion ? Spin- column or gel purification is important to get rid of residual DNA polymerase and nucleotides, in order to prevent filling of the cohesive ends generated by the restriction enzymes.
I"m cloning the EAF1 gene in pet16b vector, but I don"t know if the fragment is toxic or not. the fragment is about 255bp.Did someone know if it"s toxic?
Pierre Béguin I've also purified the fragment even after fragment digestion by running a gel and cut it again.
Hanna Alalam I use ndeI and XhoI enzymes and I've also assure that the base between end and restriction site be at least 7 .
Dear Abire,
you mentioned that the vector seems not to be the problem, because you tested it other fragments. Did these other fragments also have the same restriction enzymes as the one you are cloning currently and do you see a different running behavior of your vector after digestion, means, do you know for sure that your restriction enzymes successfully cleaved the vector? Some restriction enzymes are methylation sensitive, which would of course lead to a problem of all vectors you tested. Which restriction enzymes do you use?
@jan-Hendrik Heilers i've tested the vector with the same restrictions enzymes with others cloning of the same enzyme and it worked.the vector don't have problem because the same restrictions sites work with others fragments in which i used the same enzyme.
The problem seems to be your PCR product and based on all of the controls you have done my guess is that one or both of the enzymes is not cutting. You could try making an intermediate plasmid by inserting your PCR product without cutting into pGEM-T, isolating the recombinant plasmid and then cutting with your two enzymes. If you get the expected size restriction fragment from the digest you know then ends are right so isolate it from the gel and use that to insert into your desired plasmid. It's more work to go this way but you have tried everything else.
Kehinde Oyeniran I've even tried differents ratios but still nothing
I would try cloning into an intermediate vector and then subclone into pet16b as suggested by Mark Kainz.
Dear Abire,
As you mentioned the vector and insert were double-digested with Nde1 and Xho1. the next step is ligation. Then, you can run the mixture of digested insert and vector before/after ligation on agarose gel. you will see smear in the case of ligation lane. this smear will contain oligermization of insert and vector (insert-insert ligation, insert-vector ligation, and vector -vector ligation). this is to confirm that your insert and vector are double digested.this smear is shown in lane 3 and 4 in the attached image.
Hi Albire
I suggest you first clone your fragment into a TA vector like PTZ R/T vector and then cut it by your restriction enzymes. this way make you sure that fragments are digested.
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