IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/05/26

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5/26/10

Bootcamp Day 3

No growth observed on plates transformed with ligation products from yesterday. Re-plated 12M and 12O constructs as follows:

</table Plates resulted in lawns on LB plate but no CAP growth. PCR cleanup of digestion product (Epoch kit) leaving 4 ul to run on gel. Eluted into 30 ul elution buffer. Ligation
250 ul LB only
250 ul CAP
150 ul CAP
50 ul CAP
H2O 11 ul
Ligase Buffer 2 ul
12M or 12O 2 ul
Plasmid 2 ul
18C (promoter) 2 ul
Ligase 1 ul

Incubate 10 minutes RT Inactivate 20 minutes 80°C

NB: used 5X Invitrogen ligase buffer with NEB ligase instead of 10X NEB ligase buffer which may be a problem...

Transformation

100 ul competent cells, 20 ul ligation product

electroporate

add 100 ul LB broth

37°C 1 hour recovery.

Plating

For both 12O and 12M:

100 ul on CAP

100 ul on LB

50 ul on CAP

50 ul on LB



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