User:Chia-Hung Tai/Notebook/SBB11/Entry Base

Entry 1: 2/15/11

Prepared for PCR for pBca1766-sbb1141 and pBjh1601KC-sbb1116
-pBca1766-sbb1141
--Phusion is needed since the template is a plasmid DNA
--Instead of adding 3.3 uM 10x of the Expand Buffer, make it 5x by adding 6.6 uM
--Set up the PCR tubes following the protocol

-pBjh1601KC-sbb1116
--Dilute the oligos
--Add 363 uL of water (10 times the concentration) to cht002R
--Add 268 uL of water (10 times the concentration) to cht002F
--Take 1 uL of the mixture for each oligo and add 9 microL of water to get 10 uL
--Set up the PCR tubes following the protocol

Entry 2: 2/17/11

Prepare the PCR products in the analytical gel (since my parts does not require SOEing)
-Start out with 2 0.5 mL tubes
-Add 5 uL of loading dye buffer into each
-Add 2 uL of sbb1141 and sbb1116 PCR product into the respective tubes

Zymo Cleanup (Regular)
-Add 180 uL of Zymo ADB buffer (brown bottle) to a 33 uL reaction.
-Transfer into the Zymo column
-Spin for 30 sec in centrifuge and discard waste. (ALWAYS PUT ON LID!!!)
-Add 200 uL of Zymo Wash buffer
-Spin for 30 sec again and discard waste.
-Add 200 uL of Zymo Wash buffer for second wash
-Spin for 30 sec in centrifuge, discard waste.
-spin for 90 seconds at full speed to dry the column.
-Elute with water into a fresh Eppendorf tube, use the same volume of water as the volume of the original reaction
--Add 33 uL of water to Zymo column
--Spin for 60 sec for water to elute into Eppendorf tube

Stored products in Box B

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