2062 pEC-Mxe-gyrA
Cloning of 2062 pEC-Mxe-gyrA
[edit]Purpose: Cloning of2062 pEC-Mxe-gyrA==
Date: 2014-2----
Notebook:0
Page:
Clone ID:2062
Experimenters: shanyi
Results
[edit]CLONING
[edit]1. Gene synthesis using
Mxe_gyrA_F1 (1 ul of 10 uM)
tcgacAAGCTTGCGGCCGCActcgagggctcttcctgcatcTGTggagatgcactGgttgccctGccGgagggcgagt
Mxe_gyrA_F2 (1 ul of 1 uM)
gttgccctGccGgagggcgagtcggtacgcatcgccgacatcgtgccgggtgcgcggccGaacagtgacaacgcca
Mxe_gyrA_R1 (1 ul of 10 uM)
(G aacagtgacaacgccatcgacctgaaagtccttgaccggcatggcaatccG gtgctcgc cgaccggc)
gccggtcggcgagcacCggattgccatgccggtcaaggactttcaggtcgatggcgttgtcactgttC
2. PCR fragments using 1126 pTXB1 as template
Fragment 1
Mxe_gyrA_F3
tccG gtgctcgc cgaccggctgttccac tGc ggcgagca
Mxe_gyrA_R2
(gaaaa ccG gaatttgcg ccG a caacctacacagtcggcgt)
acgccgactgtgtaggttgtCggcgcaaattcCggttttc
Fragment 2
Mxe_gyrA_F4
G a caacctacacagtcggcgtccctggactggtgcgtttcttggaagcacac cac cgTgac
Mxe_gyrA_R3
(caccggtctgaactcaggcctcggtacc tacgtac)
gtacgtaggtaccgaggcctgagttcagaccggtg
3. Gel purify, join 3 fragments by overlap extension PCR
Mxe_gyrA_F1 + Mxe_gyrA_R3
4.digest with Kpn1 + HindIII, clone into Kpn1 + HindIII digested vector pEC
Colony Screening
[edit]Date: 2014_2_14
Make miniprep, digestion with EcoR1 + HindIII
sent 2062-1 to sequence,one extra G
Cloning of fixing 2062 pEC-Mxe-gyrA
[edit]Purpose: Cloning of2062 pEC-Mxe-gyrA==
Date: 2014-2----
Notebook:0
Page:
Clone ID:2062
Experimenters: shanyi
Results
[edit]CLONING
[edit]Fix mutation >2062 clone #1
Mxe-gyrA-fix-F
gtcgactgtgca ggt ttC gcc cgT ggC aaa ccg gaa ttt gcgccgac
Mxe-gyrA-fix-R
gtcggcgcaaattccggtttGccAcgggcGaaacctgcacagtcgac
join two fragments by pcr
.digest with Kpn1 + HindIII, clone into Kpn1 + HindIII digested vector pEC
Colony Screening
[edit]Date: 2014_2_
Make miniprep, digestion with EcoR1 + HindIII
sent 2062fix-1to sequence, one mutation
Cloning of fixing 2062fix pEC-Mxe-gyrA
[edit]Purpose: Cloning of2062 pEC-Mxe-gyrA==
Date: 2014-3----
Notebook:0
Page:
Clone ID:2062
Experimenters: shanyi
Results
[edit]CLONING
[edit]Fix mutation >2062 fixclone #1
Mxe_gyrA_F1 (1 ul of 10 uM)
tcgacAAGCTTGCGGCCGCActcgagggctcttcctgcatcTGTggagatgcactGgttgccctGccGgagggcgagt
Mxe_gyrA_R2
(gaaaa ccG gaatttgcg ccG a caacctacacagtcggcgt)
acgccgactgtgtaggttgtCggcgcaaattcCggttttc
Fragment 2
Mxe_gyrA_F4
G a caacctacacagtcggcgtccctggactggtgcgtttcttggaagcacac cac cgTgac
Mxe_gyrA_R3
(caccggtctgaactcaggcctcggtacc tacgtac)
gtacgtaggtaccgaggcctgagttcagaccggtg
join two fragments by pcr
.digest with Kpn1 + HindIII, clone into Kpn1 + HindIII digested vector pEC
Colony Screening
[edit]Date: 2014_3_
Make miniprep, digestion with EcoR1 + HindIII
sent 2062fix2-2 to sequence, good