SBB11Ntbk-JessicaWen: Difference between revisions
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= | =[[User:Jessica Wen|Jessica Wen]] 14:05, 15 February 2011 (EST)= | ||
Cloning by PCR of the following construction files | |||
Part sbb1112 {P_cspI}<br> | |||
PCR jwsbb1112F/ss37f on MG1655 (506bp, EcoRI/BamHI)<br> | |||
Sub into pBjh1601KC-Bca1144#5 (EcoRI/BamHI, 3131+910, L)<br> | |||
Product is pBjh1601KC-sb1112 {P_cspI}<br> | |||
------------------------------------<br> | |||
jwsbb1112F Forward Construction of P_cspI promoter part ccaaaGAATTCatgAGATCTATGAATCTTTAAGAGATAAAAAGC<br> | |||
ss37f BglBrick basic part cloning of cspI promoter tttggGGATCCgccatctttcggcgtgatgaaacc<br> | |||
Part sbb1137 {P_yciG}<br> | |||
PCR ss64r/ss64f on MG1655 gen. (616bp, EcoRI/BamHI)<br> | |||
Sub into pBjh1601KC-Bca1144#5 (EcoRI/BamHI, 3131+910, L)<br> | |||
Product is pBjh1601KC-sbb1137 {P_yciG}<br> | |||
------------------------------------------------------ | |||
ss64fReverse Cloning of P_yciG tttggGGATCCgtcggatgccttctcacggtcttcgg<br> | |||
ss64rForward Cloning of P_yciG aaaccGAATTCatgAGATCTagcagcgattgatgcaggagctgcg<br> | |||
Part sbb1123 {P_rfaH}<br> | |||
PCR ss49r/ss49f on MG1655 gen. (421bp, EcoRI/BamHI)<br> | |||
Sub into pBjh1601KC-Bca1144#5 (EcoRI/BamHI, 3131+910, L)<br> | |||
Product is pBjh1601KC-sbb1123 {P_rfaH}<br> | |||
------------------------------------------------------<br> | |||
ss49fReverse Cloning of P_rfaH tttggGGATCCgcatcactgactgcagtacgttttccacgcacg<br> | |||
ss49rForward Cloning of P_rfaH aaaccGAATTCatgAGATCTgcgtgatacgttttagctcaccctg<br> | |||
'''Procedure'''<br> | |||
If the oligo is not already in 10uM concentration make an oligo dilution of:<br> | |||
9uL water<br> | |||
100uL uM oligo<br> | |||
Make one PCR reaction tube of the following for each part:<br> | |||
24uL ddH2O<br> | |||
3.3uL 10x Expand Buffer "2"<br> | |||
3.3uL dNTPs (2mM in each)<br> | |||
1uL Oligo 1, 10uM<br> | |||
1uL Oligo 2, 10uM<br> | |||
0.5uL Expand polymerase "1"<br> | |||
0.5uL Template DNA<br> | |||
==[[User:Jessica Wen|Jessica Wen]] 15:08, 10 February 2011 (EST)== | ==[[User:Jessica Wen|Jessica Wen]] 15:08, 10 February 2011 (EST)== | ||
Revision as of 12:05, 15 February 2011
Jessica Wen 14:05, 15 February 2011 (EST)
Cloning by PCR of the following construction files
Part sbb1112 {P_cspI}
PCR jwsbb1112F/ss37f on MG1655 (506bp, EcoRI/BamHI)
Sub into pBjh1601KC-Bca1144#5 (EcoRI/BamHI, 3131+910, L)
Product is pBjh1601KC-sb1112 {P_cspI}
jwsbb1112F Forward Construction of P_cspI promoter part ccaaaGAATTCatgAGATCTATGAATCTTTAAGAGATAAAAAGC
ss37f BglBrick basic part cloning of cspI promoter tttggGGATCCgccatctttcggcgtgatgaaacc
Part sbb1137 {P_yciG}
PCR ss64r/ss64f on MG1655 gen. (616bp, EcoRI/BamHI)
Sub into pBjh1601KC-Bca1144#5 (EcoRI/BamHI, 3131+910, L)
Product is pBjh1601KC-sbb1137 {P_yciG}
ss64fReverse Cloning of P_yciG tttggGGATCCgtcggatgccttctcacggtcttcgg
ss64rForward Cloning of P_yciG aaaccGAATTCatgAGATCTagcagcgattgatgcaggagctgcg
Part sbb1123 {P_rfaH}
PCR ss49r/ss49f on MG1655 gen. (421bp, EcoRI/BamHI)
Sub into pBjh1601KC-Bca1144#5 (EcoRI/BamHI, 3131+910, L)
Product is pBjh1601KC-sbb1123 {P_rfaH}
ss49fReverse Cloning of P_rfaH tttggGGATCCgcatcactgactgcagtacgttttccacgcacg
ss49rForward Cloning of P_rfaH aaaccGAATTCatgAGATCTgcgtgatacgttttagctcaccctg
Procedure
If the oligo is not already in 10uM concentration make an oligo dilution of:
9uL water
100uL uM oligo
Make one PCR reaction tube of the following for each part:
24uL ddH2O
3.3uL 10x Expand Buffer "2"
3.3uL dNTPs (2mM in each)
1uL Oligo 1, 10uM
1uL Oligo 2, 10uM
0.5uL Expand polymerase "1"
0.5uL Template DNA
