Editing 1283 pDB.His.MBP.Ent

== '''Cloning of 1283 pDB.His.MBP.Ent'''  ==

'''Purpose:''' Cloning of 1283 pDB.His.MBP.Ent 

'''Date: '''July 31, 2012<br> 

'''Notebook:&nbsp;''' 

'''Page:''' 

'''Clone ID: '''1283 

'''Experimenters: '''Jennifer Truong 

== '''Results'''  ==

=== Cloning  ===

Date:&nbsp;July 31, 2012<br> 

[[Image:0731 Amp.JPG]]<br> 

PCR MBP from pMAL-p5E using<br>1 ul of 10 uM MBP-Xba1-F1 cgatcgTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATAatgggcagcagccatc<br>1 ul of 1 uM MBP-F2 GATATAatgggcagcagccatcatcatcatcatcacATGAAAATCGAAGAAGGTAAAC <br>1 ul of 10 uM MBP-Nde1-R gtgctatCATATGGCCCATGGACGGTACCTTGTCATCGTCATCCCCGAGGTTGTTGTTATTGTTATTGTTG 

Digest PCR product with Xba1 + Nde1, ligate to Xba1 + Nde1 digested vector pDB.His.BMP, V14<br> 

See lane 2, expected size ~1400 bp<br> 

Cut out band and digested <br> 

[[Image:1283 Digestion.JPG]]<br> 

Digest PCR product with Xba1 + Nde1 (lane 1), ligate to Xba1 + Nde1 digested vector pDB.His.BMP, V14 (lane 2)&nbsp;<br> 

Cut out bands, purify and ligate together overnight 

Date:&nbsp;August 7, 2012<br> 

[[Image:1283 0807 Amp.JPG]]<br> 

Sequencing came back contaminated, started all over<br> 

See lanes 1 and 2<br> 

PCR MBP from pMAL-p5E using<br>1 ul of 10 uM MBP-Xba1-F1 cgatcgTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATAatgggcagcagccatc<br>1 ul of 1 uM MBP-F2 GATATAatgggcagcagccatcatcatcatcatcacATGAAAATCGAAGAAGGTAAAC<br>1 ul of 10 uM MBP-Nde1-R gtgctatCATATGGCCCATGGACGGTACCTTGTCATCGTCATCCCCGAGGTTGTTGTTATTGTTATTGTTG 

Digest PCR product with Xba1 + Nde1, ligate to Xba1 + Nde1 digested vector pDB.His.BMP, V14 

See lane 2, expected size ~1400 bp 

Cut out band and digested <br> 

[[Image:1283 0810 Digestion.JPG]]<br> 

Digest PCR product with Xba1 + Nde1 (lane 1), ligate to Xba1 + Nde1 digested vector pDB.His.BMP, V14 (lane 2) 

Cut out bands, purify and ligate together overnight <br> 

Date:&nbsp;August 9, 2012 

Digestion was messed up from earlier because of the gel, started all over 

[[Image:1283 0809 Amp.JPG]] 

Cut out lanes 1 and 2 for digestion, save 3 and 4 in box just incase something happens 

Date:&nbsp;August 17, 2012<br> 

[[Image:1283 0817 Digestion.JPG]]<br> 

Digest PCR product with Xba1 + Nde1 (lane 7), ligate to Xba1 + Nde1 digested vector pDB.His.BMP, V14 (lane 8) 

Cut out bands, purify and ligate together overnight <br> 

=== <u></u>Colony Screening  ===

Date:&nbsp;August 3, 2012<br> 

[[Image:1283 Screening.JPG]]<br> 

Digested MiniPrep plasmids with RE Xba1 +&nbsp;Nde1 in lanes 1 - 6 (samples 1-6 respectively)<br> 

Digested sample 1 with Xba1 +&nbsp;Nco1 in lane 8, and digested sample 1 with Xba1 +&nbsp;Kpn1 in lane 9 

All correct sizes, send sample 1 to sequencing

Clones <br><br> 

'''These are contaminated clones''' 
Date:&nbsp; 2012

[[Image:1283_0820_Screening.JPG]]

Clones 2, 3, 4, 5 and 6 may be correct



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

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== '''Conclusion'''  ==

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[[Category:Cloning]]