zfatima's Content

#157109 Double strand cdna synthysis

Posted by zfatima on 28 June 2013 - 02:07 PM in Molecular Cloning

- okay thank you.

#157023 Double strand cdna synthysis

Posted by zfatima on 27 June 2013 - 11:37 AM in Molecular Cloning

gene specific? yep have been using them to synthesize first strand cdna.

#156970 Double strand cdna synthysis

Posted by zfatima on 26 June 2013 - 10:24 AM in Molecular Cloning

- 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!

#156868 TA Cloning of ds cDNA

Posted by zfatima on 24 June 2013 - 06:42 AM in Molecular Cloning

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

#156859 TA Cloning of ds cDNA

Posted by zfatima on 24 June 2013 - 12:36 AM in Molecular Cloning

- 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.

#156845 TA Cloning of ds cDNA

Posted by zfatima on 23 June 2013 - 12:41 AM in Molecular Cloning

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).

#156792 TA Cloning of ds cDNA

Posted by zfatima on 21 June 2013 - 02:24 AM in Molecular Cloning

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?

Google Sign in options