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Following the RE digest (NotI- 5'overhang) of a vector to isolate the fragments of interest I purified the inserts using electrophoresis (1%TAE gel). Insert sizes are 250bp, 290bp and 600bp. The insert appeared as a clean single band on the gel (visualised under low UV exposure). After purification (QIAEX II kit) I set up a T4 poly fill in step as follows:
27ul dna (approx 400ng total)
3.5ul 10x buffer
1ul 10mM dNTPs
1ul T4 DNA polymerase (Promega)
2.5ul H2O
35ul total
Incubated @37 degrees, 10mins (Promega protocol specifies 5mins)
I've then re-purified the insert with gel electrophoresis and instead of seeing one discrete band what I see is a fat band/multiple bands ~100bp wide. This happened with all 3 inserts modified. At first I assumed that the T4 polymerase had munched up the ends of my fragment but the expected size of the fragment was actually at the front of the band... so everything else over the ~100bp is larger than it should be
.I've had this happened twice so decided to press on and try the ligation anyway... and surprise surprise the ligation/transformation didn't work!
Following the attempted ligation/transformation I got zero colonies across all plates. (I didn't perform a +ve control, just a -ve (blunt/phosphatased vector only) control). I have had the vector transform and select successfully before so I'm sure it's an insert problem. I've abandoned the blunt end ligation in favor of a sticky end ligation with the use of a linker (PCR generated). I'm just curious if anyone would know what might cause the appearance of larger products after T4 DNA polymerase modification?
