IGEM:MIT/2006/Notebook/2006-7-26

Silent mutation to eliminate PstI site in pchA

• Diluted pchA mutation primers to 25μM
• did a PNK step on the primers (they're in little tubes in the freezer, if we want to re-do mutation without re-doing PNK)
• Used pchBA miniprep + primers & followed the stratagene site-directed mutagenesis protocol
• did a pcr cleanup after digesting old template dna with DpnI

Miniprep B0015

Did 3 minipreps of the terminator for general use

Digest R0040 and B0030(2)

Digested R0040 with E,S and B0030 with E,X
(We want to hook the promoter and RBS to each other)

Digest B0030(2) and CDS+Terminator

Cut B0030(2) with SP and CDS+Term with XP to hook up the two

• will cut B0030(2) tomorrow b/c ran out of miniprep

Digest ATF1 for hooking up to B0030(2)

ATF1 cut with EX, cut again with P to hook up to to SP cut B0030(2)

Cut B0015 with EX

Will hook up later

Cut backbone with EP

EP backbone for inserting the SPP

LIGATIONS

1. promoter R0040 + rbs B0030
2. promoter R0040 + rbs B0032
3. stationary phase promoter osmY + backbone pSB1A3-1

Transformations

1. ROO40+B0030(amp backbone) on AMP plate
2. ROO40+BOO32(amp backbone) on AMP plate
3. osmY promoter + pSB1A3-1 on AMP plate
4. pchBA w/ site mutation to eliminate PstI site in pchA on AMP plate
5. pUC19 on AMP plate

Liquid Cultures

End of the day liquid cultures for future use: ATF1, R0040, B0030(2)