My PCR-product had been generated by Phusion. So I had to create the required 3' A-overhangs needed for the TOPO.
So I did the following:
- 10ul DreamTaq buffer
- 1ul 10mM dATP
- 5ul of the PCR-product (electrophoresis validated it and I had purified it previously to get rid of Phusion, primers etc)
- 0.2ul ( = 1unit) of DreamTaq Polymerase (5U/ul)
Incubated the solution at 72C, for 8-10min. No cycle/shaking thermo-mixer.
Placed sample immediately on ice and did the TOPO:
- 1ul of above sample (now with hopefully 3' A-overhangs)
- 1ul Salt Solution
- Water up to 5ul
- 1ul TOPO vector (pCR4 TOPO)
Kit is from Invitrogen
Incubate for 5min at 22C
Place on ice and do the transformation with DH5 competent cells
Plates 50ul on one plate, and remaining volume on another plate
Incubated at 37C but I got no growth the day after. Nothing!
Also, what is the maximum volume that can be used when plating competent bacteria on agar selection-plates?
I took 50ul on on plate which was a good volume, but the remaining volume is close to 300ul. And thus the plates get really really wet so to say.