SBB12Ntbk-JonWright

Jon Wright 2/21/2012

Set up and ran gel on PCA2. Lane 8 below:

Small Frag Xymo on PCA2

Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction.
Transfer into the Zymo column (small clear guys)
Add 500uL of Ethanol and pipette up and down to mix
spin through (15s), discard waste.
Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol)
spin through (15s), discard waste.
Add 200 uL of Zymo Wash Buffer
spin through, discard waste.
spin for 90 seconds, full speed to dry.
elute with water (spin 60s) into a fresh Eppendorf tube

Digested PCA2 with Nhe, Bam

 50uL eluted DNA
 5.7uL NEB Buffer 2
 0.5uL NheI
 0.5uL BamHI

Small Frag Xymo on the gel purification product from last time (since the promoter part is considered a small one).

Jon Wright 2/17/2012

Small Frag Xymo on PCA product

Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction.
Transfer into the Zymo column (small clear guys)
Add 500uL of Ethanol and pipette up and down to mix
spin through (15s), discard waste.
Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol)
spin through (15s), discard waste.
Add 200 uL of Zymo Wash Buffer
spin through, discard waste.
spin for 90 seconds, full speed to dry.
elute with water (spin 60s) into a fresh Eppendorf tube

PCA2 on PCA product

1 ul o1
1 ul o2
1 ul PCA1
.5 ul phusion
10 ul 5x phusion buffer
5 ul 2mM dNTPs
32.5 ul H2O

Small Frag Xymo on ca998/jw000R product (see above) Regular Xymo on G00101/jw000F product

Add 180 uL of Zymo ADB buffer (brown bottle) to a 33uL or 50uL reaction.
Transfer into the Zymo column (small clear guys)
spin through, discard waste.
Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol)
spin through, discard waste.
Add 200 uL of Zymo Wash Buffer
spin through, discard waste.
spin for 90 seconds, full speed to dry.
elute with water into a fresh Eppendorf tube, use the same volume of water as the volume of the original reaction

Ran gel on ca998/jw000R and G00101/jw000F - lanes 1 and 2, below

Cut bands out of Gel and suspended together in ADB Buffer

Jon Wright 2/16/2012

Diluted JW000R and JW000F to 10uM

Set up PCRs:

24uL ddH2O
3.3uL 10x Expand Buffer "2"
3.3uL dNTPs (2mM in each)
1uL ca998, 10uM
1uL jw000R, 10uM
0.5uL Expand polymerase "1"
0.5uL Plasmid jtk2081

24uL ddH2O
3.3uL 10x Expand Buffer "2"
3.3uL dNTPs (2mM in each)
1uL G00101, 10uM
1uL JW000F, 10uM
0.5uL Expand polymerase "1"
0.5uL Plasmid 3346

Set up PCA:

38 uL ddH2O
5 ul 10x expand buffer
5 ul 2mM dNTPs
0.33 ul o1
0.33 ul o2
o.33 ul o9
0.75 ul Expand polymerase