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

Entry 3: 2/22/11

According to Chris, we used the wrong wash buffer during the Zymo cleanup (NEED A4 instead of AW). This messed up our product. Chris helped us to redo the PCR products. :D

EcoRI/BamHI Digest of PCR Products
-We need to digest our PCR products

-Prepare 2 PCR tubes, one with sbb1141 product and the other with sbb1116 product
-Add the following to each tubes:
--8 uL of the PCR product
--1 uL of NEB Buffer 2
--0.5 uL EcoRI
--0.5 uL BamHI

-Incubate the tubes at 37 degree C in the thermocycler for 1 hour.
-Run a gel

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