Berk2010-Christoph

Christoph Neyer 15:54, 7 June 2010 (EDT)

Eco/Bam digest of parts: Bjh2245 (LifeACT), Bjh1882 (RFP), and BCA1091 (Ptet).
Gel purified Bjh1882 and Bjh2245.
Small fragment zymo cleanup of BCA1091.

Christoph Neyer 16:35, 7 June 2010 (EDT)

Couldn't see fragments. Turns out Bjh2245 is 97bp and Bjh1882 is ~680bps.
Re-cut Bjh2245 and Bjh1882.
Small fragment zymo cleanup of Bjh2245 digest.


Gel purified Bjh1882.

Christoph Neyer 19:30, 7 June 2010 (EDT)

Bjh1882 Vectors: AC
Bjh2245 Vectors: KA
Bca1091 Vectors: CK
pMLL4-AC
pMLL5-CK
pMLL6-KA
pMLL7-AK
pMLL8-KC
pMLL9-CA
Bjh1882+pMLL4-AC = Lefty
Bjh2245+pMLL6-KA = Righty
Bca1091+pMLL5-CK = Lefty
Ligated isolated Bjh1882 and Bca1091 into above vectors. Transformed into Lefty strains.
Need to ligate Bjh2245 (Missing pMLL6, need to digest).
TO DO:

• Digest pMLL6 with Eco/Bam and gel purify. Then ligate with Bjh2245.
  
• Check transformed Bjh1882 and Bca1091.
• Transform ligated Bjh2245+pMLL6 into Righty strain

Christoph Neyer 14:04, 8 June 2010 (EDT)

• Digested pMLL6 w/ Eco/Bam and gel purified.
• Colony PCR of transformed pMLL4-Bjh1882 and pMLL5-Bca1091:
  

PCR Block 1882 and 1091 #1	'	'	'	'
1882		1091 #1		1091 #2
A		A		A
B		B		B
C		C		C
D		D		D

Christoph Neyer 16:59, 8 June 2010 (EDT)

• Ligated pMLL6 Eco/Bam digest with part Bjh2245.
• Ran gel of colony PCR pMLL4-Bjh1882 and pMll5-Bca1091:

ladder

1882a

b

c

d

1901a(1)

b

c

d

ladder

1901a(2)

b

c

d

'

'

'

'

'

'

• Repicking colonies since Colony PCR appeared contaminated.

PCR Block 1882 and 1091 #2	'	'	'	'
1882		1091 #1		1091 #2
A		A		A
B		B		B
C		C		C
D		D		D