6 replies to this topic

[zfatima]

Hi, im trying to clone viral ds cDNA into TA. i reverse transcribe by using super script III and then make double strand cDNA by using Invitrogen's SuperScript Double Strand cDNA synthesis kit. after doing poly-A tailing of the template with taq i went with ligation and transformation but to no avail. any help in this regard, please?

[bob1]

Which TA cloning vector did you use and what conditions did you try?

[zfatima]

i use pcr2.1 vector and did ligation at 22C for 4 hours and at 14C for 16 hour(just to try the best ligation time for my vector and insert).

[bob1]

Ok, and ratios of vector to insert - basically we need some information to work on, protocols and the like, otherwise there are heaps of things that could be going wrong.

[zfatima]

- sure, insert is structural portion of dengue virus which is almost 2.4kb long. i use 1:1 ratio of vector:insert. transformation is done in top10 by heat-shock method and i use ampicilin and tetracycline selection. my colony pcr gave results with gene specific and m13 rpimers. but after miniprep i got amplicon which is almost 100bp more than the required amplicon. and in the end my sequencing gave negative results. next time i ran my ligation product but it was a single band of vector, meaning i didnt get any ligation.

[bob1]

OK, may I ask why double selection? you should only need one for most plasmids.

The positive PCR was from the ligation mix spread on the plate during the transformation. You may have had some ligation happening but it didn't transform well, which could be a sign of a toxic gene product.

I take it the ratios you used were molar ratios? If so, try a few different one, usually in the range of (insert:vector) 3:1 - 10:1 works well.

Make sure that your A tailing is done fresh before the ligation, I find that storing it, even overnight, results in loss of the A tail somehow.

[zfatima]

- okay i'll try with increasing ratio of insert:vector. thank you for this discussion.