Here are the details.
1. I did PCR for PDAT, got a very specific and sharp band, eluted it out of gel using QIAGEN gel purification kit.
P.S. For PDAT, i used primers having restriction sites in them, AscI (on forward primer) and HindIII (Reverse primer), in order to get a double digest fragment (later) with only my PDAT gene inside the flanking sites.
2. Similarly i did minipreparation for pCAMBIA0390, ran it on gel, again got one very sharp and intense band ,eluted it out of gel using QIAGEN gel purification kit.
3. I double digested PDAT and pCAMBIA0390 (individually, in different tubes) with AscI and HindIII for overnight (~16-18 hours) at 37C
4. I estimated concentrations of my insert and vector after digest , which came out to be 113 ng/ul (insert; PDAT) and 300 ng/ul (vector; pCAMBIA0390).
Following concentrations i used for ligation (T4 DNA ligase; NEB):
reaction volume = 20ul
Molar ratio 1 (Vector) : 3 (insert)
10x ligation buffer - 4ul
T4 DNA ligase (400,000 U/ml) - 1ul
PDAT (insert) - 9ul [molar concentration - 0.1323 pM / ul]
PCAMBIA0390 (vector) - 6ul [molar concentration - 0.0667 pM / ul]
5. Ligation was set at 16C for ~36-48 hours.
6. Competent DH5a cells (from Invitrogen) were used. Following procedure was used:
a) Cells thawed on ice for 10 minutes
10ul of ligation mixture was added to 50ul of competent cells, and aspirated very gently to mix everything.
c) mixture kept on ice for 30 minutes
d) Heat shock at 42C for 50 seconds, and immediately kept on ice for 2 minutes.
e) 1ml LB (without Ab) added to cells gently and mixed by inverting before incubation at 37C for 2 hours.
f) 20ul and 200ul (on two separate plates) was plated on LB+Kan plates and incubated at 37C for 16-20 hours.
7. Next day after~16 hours i got 9 colonies (which looked really nice, as they were not small and looked healthy)
8. I picked each colony, resuspended cells in 10ul nuclease free water, and used 2ul of these resuspended cells for Colony PCR.
9. In PCR, i did not get any amplification for any colony, as i had run control (PDAT PCR ; done 2 weeks back) to check size of insert in my colonies.
I repeated all this twice till now, with no success.Last time i tried, i used ~80ng (total) DNA in ligation mixture (20ul), and this time i used almost 1.4ug DNA (20 ul reaction).
P.S. for T4 DNA ligase (NEB), they say that total concentration of DNA in ligation mixture should be between 1-10ng / ul i.e. if my reaction mixture is 20ul, total DNA concentration in mixture should be between 20ng to 200 ng. Could this be a problem? As last time i got 1 or 2 colonies, but without insert, whereas on second attempt i got 8-9 colonies without insert.
I have attached a file, showing primers which i used for checking my transformed colonies for insert. With Primers A and A' from vector sequence (not including the insert, but flanking just outside the insert); Primer B is used to test the orientation of insert.
Following two combinations of primers were used: A+A' and B+A'; none of them gave any amplification.
One more thing, is it true that if i am using 2 different Restriction enzymes (AscI and HindIII) for my vector, so it should not self ligate on its own, and also vector has bacterial Ab resistance gene (Kan), and if colonies are appearing on my plate, it means some insert has gone inside (as vector itself cannot self ligate). Both AscI and HindIII generate different and incompatible 5' overhangs.
Kindly let me know if i need to improve on something, or where am i going wrong. I would really appreciate your comments and suggestions.