I am trying to make jute genomic library to screen for colonies containing simple sequence repeats ( eg. AGAGAGAGAG, CTCTCTCTCTCT)
For this purpose the genomic DNA is partially digested. In a single digestion reaction, I incubate the jute genomic DNA (10ug) with SauIIIAI (4U) in a 30 ul reaction for 6 hrs. The digested DNA is run on a gel 1 % agarose gel and the DNA smear between 500bp - 1000 bp is cut. The DNA from the gel is extracted using Eppendorf's Gel Clean-up Kit and finally eluted in a 20ul volume.
The SauIIIAI cut fractionated genomic DNA is then ligated it with commercial BamHI cut BAP treated pUC118 ( from takara ). 100ng of cut vector + 600 ng of fractionated DNA + 1U Ligase, in a 10ul reaction. The ligation reaction is kept O/N at 2-4 Celsius.
2ul of the ligation mixture is mixed 100 ul of electrocompetent DH5a E.coli cells (1xe10 cells/ml), kept on ice for 1-2 minute and then electroporated at 25000 V/cm at 1.5ms time constant.
Following the above protocol I am only getting about 300- 500 colonies per transformation. However I have been asked to get more 10 fold the current efficiency.
Few suggestions have been given like using BSA, PEG, extra ATP during ligation? Will this solve the problem. Is there anything else to do?
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1 reply to this topic
Posted 15 July 2004 - 03:40 AM
your protocol seems fine. There must be an inhibitory agent decreasing your ligation efficiency. After eluting your dna from the gel by using the kit, ppt the extracted DNA and wash it with 70% ethanol twice. You can resuspend it back in to 20ul volume. Find the DNA conc and set your ligation.