Editing 2062 pEC-Mxe-gyrA

== '''Cloning of 2062 pEC-Mxe-gyrA'''  ==

'''Purpose:''' Cloning of2062 pEC-Mxe-gyrA'''==''' 

'''Date: '''2014-2---- 

'''Notebook:0''' 

'''Page:''' 

'''Clone ID:2062''' 

'''Experimenters:'''&nbsp; shanyi 

== '''Results'''  ==

=== CLONING  ===

1. Gene synthesis using<br>Mxe_gyrA_F1 (1 ul of 10 uM)<br>tcgacAAGCTTGCGGCCGCActcgagggctcttcctgcatcTGTggagatgcactGgttgccctGccGgagggcgagt<br>Mxe_gyrA_F2 (1 ul of 1 uM)<br>gttgccctGccGgagggcgagtcggtacgcatcgccgacatcgtgccgggtgcgcggccGaacagtgacaacgcca 

Mxe_gyrA_R1 (1 ul of 10 uM)<br>(G aacagtgacaacgccatcgacctgaaagtccttgaccggcatggcaatccG gtgctcgc cgaccggc)<br>gccggtcggcgagcacCggattgccatgccggtcaaggactttcaggtcgatggcgttgtcactgttC 

2. PCR fragments using 1126 pTXB1 as template<br>Fragment 1<br>Mxe_gyrA_F3<br>tccG gtgctcgc cgaccggctgttccac tGc ggcgagca 

Mxe_gyrA_R2<br>(gaaaa ccG gaatttgcg ccG a caacctacacagtcggcgt)<br>acgccgactgtgtaggttgtCggcgcaaattcCggttttc 

Fragment 2<br>Mxe_gyrA_F4<br>G a caacctacacagtcggcgtccctggactggtgcgtttcttggaagcacac cac cgTgac 

Mxe_gyrA_R3<br>(caccggtctgaactcaggcctcggtacc tacgtac)<br>gtacgtaggtaccgaggcctgagttcagaccggtg 

[[Image:2028 2062firstpcr 2068pcr.JPG]]<br> 

3. Gel purify, join 3 fragments by overlap extension PCR 

Mxe_gyrA_F1 + Mxe_gyrA_R3 

[[Image:2062 2028pcr.JPG]]<br> 

4.digest with Kpn1 + HindIII, clone into Kpn1 + HindIII digested vector pEC 

[[Image:DIGEST FOR 2062.JPG]]<br> 

<br> 

=== Colony Screening  ===

Date: 2014_2_14 

Make miniprep, digestion with EcoR1 + HindIII <br> 

[[Image:2062 2063DIGEST.JPG]] 

sent 2062-1 to sequence,one extra G<br> 

== '''Cloning of fixing 2062 pEC-Mxe-gyrA'''  ==

'''Purpose:''' Cloning of2062 pEC-Mxe-gyrA'''==''' 

'''Date: '''2014-2---- 

'''Notebook:0''' 

'''Page:''' 

'''Clone ID:2062''' 

'''Experimenters:'''&nbsp; shanyi 

== '''Results'''  ==

=== CLONING  ===

Fix mutation &gt;2062 clone #1 

Mxe-gyrA-fix-F<br>gtcgactgtgca ggt ttC gcc cgT ggC aaa ccg gaa ttt gcgccgac<br>Mxe-gyrA-fix-R<br>gtcggcgcaaattccggtttGccAcgggcGaaacctgcacagtcgac 

[[Image:2062fixpcr1st.JPG]] 

join two fragments by pcr 

[[Image:1560fixpcr 2062fixpcr2.JPG]]<br><br> 

.digest with Kpn1 + HindIII, clone into Kpn1 + HindIII digested vector pEC 

[[Image:Digest for 1560fix 2062fix.JPG]]<br> 

<br> 

<br> 

=== Colony Screening  ===

Date: 2014_2_ 

Make miniprep, digestion with EcoR1 + HindIII <br> 

<br> 

[[Image:1560fix 2062fix 2070digest.JPG]]<br> 

sent 2062fix-1to sequence, one mutation<br> 

== '''Cloning of fixing 2062fix pEC-Mxe-gyrA'''  ==

'''Purpose:''' Cloning of2062 pEC-Mxe-gyrA'''==''' 

'''Date: '''2014-3---- 

'''Notebook:0''' 

'''Page:''' 

'''Clone ID:2062''' 

'''Experimenters:'''&nbsp; shanyi 

== '''Results'''  ==

=== CLONING  ===

Fix mutation &gt;2062 fixclone #1 

Mxe_gyrA_F1 (1 ul of 10 uM)<br>tcgacAAGCTTGCGGCCGCActcgagggctcttcctgcatcTGTggagatgcactGgttgccctGccGgagggcgagt 

Mxe_gyrA_R2<br>(gaaaa ccG gaatttgcg ccG a caacctacacagtcggcgt)<br>acgccgactgtgtaggttgtCggcgcaaattcCggttttc 

Fragment 2<br>Mxe_gyrA_F4<br>G a caacctacacagtcggcgtccctggactggtgcgtttcttggaagcacac cac cgTgac 

Mxe_gyrA_R3<br>(caccggtctgaactcaggcctcggtacc tacgtac)<br>gtacgtaggtaccgaggcctgagttcagaccggtg<br>[[Image:2062fix2pcr1.JPG]]<br> 

join two fragments by pcr 

[[Image:2132 2062fix2.JPG]]<br><br> 

.digest with Kpn1 + HindIII, clone into Kpn1 + HindIII digested vector pEC 

<br> 

<br> 

<br> 

=== Colony Screening  ===

Date: 2014_3_ 

Make miniprep, digestion with EcoR1 + HindIII <br> 

[[Image:2062fix2 2132digest.JPG]]<br> 

sent 2062fix2-2&nbsp; to sequence, good<br><br> 

<br> 

<br> 

== '''Result intepretation/Summary'''  ==

*<br>

== '''Conclusion'''  ==

[[Category:Cloning]]