NC4-dmuctraf.doc - Transformation of NC4 or D mucoroides...

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Transformation of NC4 or D. mucoroides with vectors containing the V18-Tn5 cassette (Harry MacWilliams, September 1998) (Directions are for transforming one vector) Materials: KK 2 : 17 mM potassium phosphate, pH 6,2; sterile. KK 2 agar plates. These contain 1,5% agar in KK 2 , autoclaved. These should be poured LEVEL, ie, they should be allowed to cool on a precisely horizontal surface; if stacked whiile cooling, check to see that the stacks are really straight. The plates should not be wrapped up immediately after hardening, but allowed to dry overnight. I just put the stacks of plates, closed of course, on the bench; the laminar flow hood is also OK, but it should be turned off so the plates do not dry out on one side. G100 plates: KK 2 agar plates containing 100ug/ml G418, added after the agar has cooled to 60 degrees. My G100 plates contain 20 ml of agar (pipetted, not poured); accuracy is important as the amount of agar controls the severity of the selection. 2x MOPS-phosphate. This contains 40mM MOPS and 1,4mM sodium phosphate, pH 7,1, sterilized by filtration. 1x MOPS-phosphate. The above, diluted 1:1 with sterile H 2 0. SM plates (after Sussman) spread with Klebsiella aerogenes and incubated for two days at 37 degrees. Bacterial suspension. This is made by suspending the pregrown Klebisiella aerogens in KK 2 . A 1x suspension contains the bacteria from one plate in 2,5 ml of KK 2 . A 2x suspension contains the bacteria from 2 plates in 2,5 ml KK 2 .
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Christopher Reinemann
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