2301 pR-wtSM2-NucA
Cloning of 2301 pR-wtSM2-NucA
[edit]Purpose: Cloning of2301 pR-wtSM2-NucA==
Date: 2014-8----
Notebook:0
Page:
Clone ID:2301
Experimenters: shanyi
Results
[edit]CLONING
[edit]gene synthesis
2nd fragment
PCR sm2 gene from 1680 pCold-GST-wtSM2 using this pair of oligos
2301-SM2-NucA-Nde1-F GCCgac catatgacgctcgaatccatcgac
old oligo
1 ul of 10 uM WtSM2-Xho1-R
(gagttgatgggctgcaaaaac taa ctcgag ctagca)
tgctagctcgagttagtttttgcagcccatcaactc
join 2 fragment
2301-SM2-NucA-1 TAGCACAGATCTACGTGTGGCCTGGCTGTACCATGACTGACACATTCACAACATG
1 ul of 10 uM WtSM2-Xho1-R
(gagttgatgggctgcaaaaac taa ctcgag ctagca)
tgctagctcgagttagtttttgcagcccatcaactc
digest with BglII + Xho1
Colony Screening
[edit]Date: 2014_8
cloning, grow at 30 oC to suppress expression.
expression, grow at 30 oC, induce at 37 ~ 42 oC.
Make miniprep, use BglII + Xho1 to find positive clone
send 2301-2 to sequence, it is the correct clone
Transform Ecoli K12 JM101, grow at 37oc overnight, the next morning, inoculate sevral colonies into 5ml LB, grow at 37oC overnight.
add 20 ul culture medium to 2ug plasmids DNA, incubate 30min,run gel
