from the T. brucei project

MAKING END - PROBES FROM P1 CLONES.

from Wesley, C.S., Myers, M.P. and Young M.W. (1994). Rapid sequential walking from termini of cosmid, P1 and YAC inserts. Nucl. Acids. Res. 22(3), 538-539.

Degenerative primer : 5' GTC AGT CAG TCA GAN NNN GAG 3'

1uL P1 DNA

5µL 10x PCR Buffer

0.5µL 20mM dNTPs

1µL Taq (or whatever is standard for your general use)

2µL Degenerate Primer (25ng/µL)

37.5µL H2O

These reagents should be mixed together and then started on the following PCR cycle:

95°C 30 seconds

30°C 4 minutes Ramped at 5.3°C per minute

75°C 4 minutes

Hold at 75°C whilst you add

2µL Degenerate Primer (25ng/µL)

1µL T7 OR SP6 (100ng/µL)

Then do 35 cycles of

95°C 30 seconds

52°C 1 minute

75°C 1 minute

I routinely run 5µL of the PCR product on a mini gel. Often the products are quite small, so don't use bromophenol blue as your loading buffer.

Any questions to Caroline Gerrard

        	  T7
------------------------------------------------------
Done 	     76 
Success   	66	(87% success)
 
 
 
        	  SP6
-------------------------------------------------------
Done	       50
Success         32     (64% success)