- im trying to synthesize double strand cDNA (viral) using invitrogen's superscript double strand cDNA synthesis kit but having problems which surely are related to rna concentrations. has anyone used this kit and at what rna concentrations they proceeded with the synthesis? Kindly help!
- 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.
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?