SBB11Ntbk-SuhaniVora

Suhani Vora 14:08, 15 February 2011 (EST)

Today we set up PCR reactions for our promoter parts.
-Resuspended oligos PmalK_R and SV_06 to 100 uM.
-Diluted primers to 10 uM.
-Set up PCR for cloning using Expand Polymerase
1. P_malk
2. P_nlpA
3. P_rfaQ
Thermocycler Program: 2K55

Construction Files:

PCR ss61f/P_malKR on E. coli MG1655 gDNA  (525 bp, EcoRI/BamHI)
Sub into pBjh1601KC-Bca1144               (EcoRI/BamHI, 3131+910 bp, L) 
Product is pBjh1601KC-sbb1134             {P_malK}
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ss61f	BglBrick basic part cloning of lamB promoter	AAACCGAATTCATGAGATCTATGCGGATAATGCGAGGATGCGTGCACCTG
P_malkR	BglBrick basic part cloning of lamB promoter	CTGATggatccACCTTCATGGATATCGAGATTG

Part sbb1132                       {P_nlpA}
PCR ss58r/SV_06 on MG1655 gen.              (542bp, EcoRI/BamHI)
Sub into pBjh1601KC-Bca1144#5                 (EcoRI/BamHI, 3131+910, L)
Product is pBjh1601KC-sbb1132                       {P_nlpA}
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ss58r	Forward Cloning of P_nlpA                                 aaaccGAATTCatgAGATCTgcttcccaataattgctctg
SV_06	P_nlpAR  (reverse cloning of P_nlpA BglBricks basic part) CTGATGGATCCCCGTAGATGATGTGTTGTCAG

Part sbb1121                       {P_rfaQ}
PCR ss47r/ss47f on MG1655 gen.                (1031bp, EcoRI/BamHI)
Sub into pBjh1601KC-Bca1144#5                 (EcoRI/BamHI, 3131+910, L)
Product is pBjh1601KC-sbb1121                       {P_rfaQ}
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ss47fReverse Cloning of P_rfaQ     tttggGGATCCttttcagacaaaatagggatggtgtcctg
ss47rForward Cloning of P_rfaQ     aaaccGAATTCatgAGATCTattttacgtttatgtagcgccgcaatcagg

Suhani Vora 13:24, 17 February 2011 (EST)

Ran PCR samples of P_malK, P_nlpA, P_rfaQ on analytical gel.

2ul Sample + 5 uL Loading Dye Buffer

Sample 1: P_malK (525 bp)
Sample 2: P_nlpA (542 bp)
Sample 3: P_rfaQ (1031 bp)

Cleaned with Zymo Cleanup : May have used wrong buffer

All PCR products for the class were thrown out, re-done by Prof. Anderson.

Suhani Vora 13:37, 22 February 2011 (EST)

P_nlpA PCR failed yesterday. Will have to move on without that part.

EcoRI/BamHI Digest of P_rfaQ (sbb1121) and P_malK (sbb1134)

Place in thermocycler at 37 deg for 1 hr.

Lane 3: sbb1121 (P_rfaQ) [1031 bp] Lane 4: sbb1134 (P_malK) [525 bp]