Hi Maria,

if your plan is only ligate the plasmid your protocol is ok and I have some suggestions that worked for me.

A ligation is performed using 50-100 ng total (so your choice is ok). You can eliminate the inactivation step in between the kinase and ligase. So after 30 min at 37 Celsius with the kinase, immediately add the ligase and keep it at room temperature (22 Celsius) for 2 h, finally inactive the ligation at 65 Celsius for 10 min.

Transform:

5 ul of that ligation sample + 100 ul NEB 10B cell (optional 2 ul ligation+ 40 ul NEB 10B cells ), if chemical competent place the cell vial on ice when thawed add the desired ligation volume, place the mix on ice for 5 minutes then a heat shock at 42 Celsius for 45 seconds, put back the mix on ice 3-5 min then add 200-300 ul LB (no antibiotic) and incubate it at 37 Celsius, 220 rpm for 60 min then plate 50-100 ul of the incubated culture on the LB/(desired antibiotic) dish and incubate it Overnight.

If you need more details please let me know

Noe Baruch

Thanks for the help Noe!

I wil definetely let you know if i need more help!

Hey,

I always have problems with PCR+ t4 pnk+t4 DNA ligase protocols.

I would suggest to use neb Q5 site directed mutagenesis kit to delete the fragment, it has its own pre-made reagents that works realy efficiently.

Update:

the PCR-phosphorylation-ligation seems to be working.

After the PCR reaction I've used PCR cleanUp kit.

The phosphorylation:

Water = 15 ul

T4 DNA ligase buffer x10 = 2,0 ul (Buffer with 10mM ATP BioLabs)

T4 kinase (PNK) = 1ul (NEB)

DNA (120 ng)= 2 ul 20 ul (total vol.)

Incubation at 37 C, 30 min. Heat Inactivation prior to ligation- 65°C for 20 minutes. Cool on ice. Ligation reaction: Add to phosphorylation reaction (after cooling) 1ul T4 DNA ligase (Roche)

Incubate 16°C O/N. Ligation is ready for transformation (heat-shock only).

I haven't perform ligation deactivation before the heat-shock.

Some colony were sequenced and seem to be goed.