PMBP-Kinase-Cloning
1. Check kinases in pDNR-Dual vector:
- Make sure the kinase has no internal Sal1 and BssHII/AvrII sites
- Make sure it is not a receptor kinase
2. Mix plasmid DNA in one tube
~ 1 ug kinase in pDNR-Dual
~ 0.5 ug pMBP vector
add 1.5 ul NEBuffer, add water to 15 ul
add 0.3 ul Sal1(HF) + 0.3 ul BssHII
or 0.3 ul Sal1(HF) + 0.3 ul AvrII
Incubate at 37 oC for 1 ~ 2 hours.
3. Add 2 ul (1/10 volume) of 3M Potassium Acetate (pH3).
4. Add 40 ul (2 volumes) of 100% ethanol (or 15 ul isopropanol)
5. Incubate on ice for 15 mins.
6. Spin at 13,000 RPM for 5 mins.
7. Carefully remove (aspirate off) ethanol.
8. Wash once with 30 ul 70% ethanol.
9. Carefully remove ethanol and leave pellet to air dry.
10. Add 20 ul water to dissolve DNA.
11. Add 2.5 ul ligation buffer, 0.5 ul ligase, ligate at RT for 2 hour.
12. Use entire reaction for transformation (add 100 ul Top10).
13. Spindown and plate all bacteria on LB/6%Sucrose/Kan plate.
14. Grow O/N at 37 oC.
Faster protocol (step 2 to 10 modified)
2. Digest kinase plasmid DNA
~ 1 ug kinase in pDNR-Dual
add 1.5 ul NEBuffer, add water to 15 ul
add 0.3 ul Sal1(HF) + 0.3 ul BssHII
or 0.3 ul Sal1(HF) + 0.3 ul AvrII
3. Heat Inactivate at 80 oC for 20 min on a PCR machine with hot lid on.
4. Add gel-purified predigested pMBP.
5. Add to final vol ~22 ul.
6. Follow step 11 to 14 as above.