SBB11Ntbk-JessicaWen: Difference between revisions
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==[[User:Jessica Wen|Jessica Wen]] 13:31, 17 February 2011 (EST)== | ==[[User:Jessica Wen|Jessica Wen]] 13:31, 17 February 2011 (EST)== | ||
'''Analytical Gel of PCR Products''' | '''Analytical Gel of PCR Products'''<br> | ||
'''Procedure''' | '''Procedure'''<br> | ||
In a 0.5mL tube add: | In a 0.5mL tube add:<br> | ||
2uL of PCR product | 2uL of PCR product <br> | ||
5uL of DNA Loading Buffer | 5uL of DNA Loading Buffer <br> | ||
Run Gel | Run Gel | ||
Revision as of 11:36, 17 February 2011
Jessica Wen 13:31, 17 February 2011 (EST)
Analytical Gel of PCR Products
Procedure
In a 0.5mL tube add:
2uL of PCR product
5uL of DNA Loading Buffer
Run Gel
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
