I am trying to screen a library consisting of genomic insert in pUC18 plasmids and transformed in DH5a E coli.
After lifting the colonies on nylon membrane, I am screening them with a repetitive probe of T(AG)8. I have also included a positive control (a clone consisting of T(AG)8 insert in pUC18).
I am using Church Buffer for pre-hyb and hyb @42C. And for stringency washes I am using 2xSSC 0.1% SDS @ rtp for 5 mins on the first wash followed by 0.5xSSC 0.1%SDS. @ 48C for 15 mins on the second wash.
The problem is I am not getting any signals, not even for my positive control.
Is there any suggestions? How good is Church buffer for hybridization?
What temperature should the hybridization and stringency wash should follow?
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?