Ligation worked, colonies on plate but none positive - (Oct/06/2015 )

Hi all,

I've been having huge problems cloning the 1.8kb CAGG promoter into a 9kb vector. I amplified the CAGG sequence from a plasmid to have MluI and NheI RE sites suitable for cloning into my new vector.

The PCR required some optimisation (GC buffer etc) but worked. Both vector and amplicon were sequentially digested with the enzymes (as they are not compatible), and final products gel extracted (I observed a small amount of STAR activity of NheI on the CAGG promoter).

I used T4 ligase (NEB) with 30ng vector and all the amplicon I had left (~30ng) in an overnight 16oC reaction, and also did a no insert control.

I tested the ligation reactions by PCR to confirm, using primers directed against both insert and vector, that ligation had occurred, and it had in the insert containing reaction.

I transformed into both DH5a (library efficiency) and DH10B electrocompetent cells. I obtained NO colonies on the control no insert plates, and 30-40 colonies on the DH10B plate (but none on DH5a).

So far, so good. I did a colony PCR on 20 colonies but not one was positive, and miniprep and digest did not give the correct bands (they matched the parent vector).

I am very confused by this! Background undigested/religated vector happens from time to time, but I controlled for this and this didn't happen!

Any ideas/suggestions would be hugely appreciated!

Thanks

Antony

i usually use calcium chloride to make competent cell and do heat shock at 42 degree. and get good results 

1) Did you add a 5' extension beyond the restriction site on your primers to facilitate cutting of the PCR fragments?

2) Your ligation PCR only tested one of the two ligation sites. Also, it tells little about the efficiency of the ligations.

Thanks phage

1) Yes, I had 6bp extra on both primers

2) I did a ligation pcr across both junctions and both were positive

the confusion for me comes in that I had no background on the control plate, but some on the ligation reactions that had the linear fragment added!