Hello all,
I'm trying to clone a 500 kb fungal DNA fragment, generated from RAPD PCR, into a PJET cloning vector (Thermo Scientific) that has a 98% selection capability. I am verifying the cloned insert using PCR with the prescribed primers, and I consistently obtain a positive result. However, every time I send the sample for Sanger sequencing, I receive the complete vector sequence rather than the specific cloned insert sequence that I am targeting.
Can anyone suggest why this might be happening? For further reference, I have also attached a gel picture of the insert PCR. Additionally, could a low concentration of the insert DNA after plasmid isolation (around 15 ng/µL) contribute to this issue?
Hi! So it's the size of the insert. 500Kb is very big, especially in comparison to the size of the plasmid. There is a much higher probability that the ends of the plasmid meet and ligate than the ends of the 500kb fragment. I think you need a bac kit. Good luck!
I would suggest you run a gel and where the plasmid is digested with enzymes that release the insert and alongside with one enzyme that linearizes the DNA but does not release the plasmid.
One possibility is that you have mixed DNA prep, most plasmid without insert but some plasmid with insert. So overall the sequence is parent plasmid but there is enough plasmid with insert to give you a PCR product. These two digests will show you if all the plasmid in your prep is homogenous or is a mixture.
Hi Sunit -
I have a question. Is there any insert in the multiple cloning site coming up when you sequencing your clone or is it really just empty vector (this is not completly clear to me from your describtion - however from your gel it looks like that there is an insert).
Is there any possibility that your RAPD Primer is annealing in the pJET vector itself and that you have a contamination of pJET in your PCR reaction? If a vector is used often in a certain lab even the pipettes can be a source of contamination. This is just an idea, but in that case there is the possibilty that you have cloned a part of pJET in the pJET vector itself and you might not recognizes this by simply aligning the sequences. If you haven't already I would carefully check the Multiple cloning site after sequencing to make sure that the vector is really empty. You can not see this very good on the gel (I think it is a little bit overloaded) but your vector seem to run quite high for a 3 kb vector - however I don't know the DNA ladder you used. Regarding your low DNA concentration ( I don't really think this is the issue) but sometimes you can increase the plasmid yield by using more of the antibiotic in the liqiud culture (2-3 times).
Another possibilty would be - as Michael J. Benedik suggested - is a mixed DNA Prep - if this is the problem I would retransform a very small amount of the plasmid prep and pick new colonies.
Dörthe Andrea Kesper Thankyou for your reply and suggestion. chances of vector contamination is very less because we don't use this vector in everyday experiment and about the ladder size I have used 100bp ladder.
Hi Sumit,
so you have checked that the multiple cloning site is really empty? If you are willing to share your sequencing results I might double ckeck if you are interested.
Did you use the primers provide with the kit for the PCR (to check for the insert) and for sequencing (pJET 1.2 forward and reverse sequencing primer)?
Which Primers did you use for the amplification of your fungal DNA?
Which restriction enzyme(s) did you use for digesting the plasmid?
However, if you don't like to share more details I have some additional suggestions for you:
1. Is it possible to sequence the PCR product directly? I am not a specialist in RAPD PCR - however if you used only one primer for amplification this might not be feasable.
2. Use another vector systems - I have often used the TOPO vector for cloning of PCR products and that worked usually very well.
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