Paul.xu at 18:27, 5 June 2011

2011-06-05T18:27:12Z

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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.

[[Category:Protocols]]

PMBP-Kinase-Cloning - Revision history

Paul.xu at 18:27, 5 June 2011