SBB12Ntbk-JonWright: Difference between revisions
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==[[User:SBB12Indiv-1208| Jon Wright]] 2/17/2012== | |||
Small Frag Xymo on PCA product | |||
<pre>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</pre> | |||
PCA2 on PCA product | |||
<pre>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</pre> | |||
Small Frag Xymo on ca998/jw000R product (see above) | |||
Regular Xymo on G00101/jw000F product | |||
<pre>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</pre> | |||
Ran gel on ca998/jw000R and G00101/jw000F | |||
[[Image:NEB_2-log_ladder.gif]][[Image:Gel2_from_class_2_17_2012.jpg|500px]] | |||
Cut bands out of Gel and suspended together in ADB Buffer | |||
==[[User:SBB12Indiv-1208| Jon Wright]] 2/16/2012== | ==[[User:SBB12Indiv-1208| Jon Wright]] 2/16/2012== | ||
Revision as of 18:42, 27 March 2012
~~!~~
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
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