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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 Cloning by PCR Protocol
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}
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}
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}
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 [Lane 3]: P_malK (525 bp)
Sample 2 [Lane 4]: P_nlpA (542 bp)
Sample 3 [Lane 5]: 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) using Using EcoRI/BamHI Digest Protocol

Place in thermocycler at 37 deg for 1 hr.

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

Gel was checked and cut by Prof. Anderson.

Shout out to Chris for hookin' us up with some sweet PCR.

(Gel cut out by Prof. Anderson, thanks!)

Suhani Vora 13:59, 24 February 2011 (EST)

Zymo Gel Purification of sbb1121 and sbb1134 usingZymo Gel Purification Protocol

Eluted in 8ul DDW

Happy Birthday Vinidhra Mani!!!

Suhani Vora 14:31, 1 March 2011 (EST)

Ligation of sbb1121 and sbb1134 (EcoRI/BamHI) into pBjh1601KC--Bca114(EcoRI/BamHI)

Followed Ligation of EcoRI/BamHI digests Protocol

Transformation of pBjh1601KC-sbb1121 and pBjh1601KC-sbb1134 into competent E. coli cells.

Followed Transformation by Heat-shock Protocol

Plated on Kanamycin plates

Suhani Vora 14:03, 3 March 2011 (EST)

Plates look fine, transformation plates for both rxn. with ssb1121 and ssb1134 had over 100 single colonies

Colonies were picked for us, 4 cultures of each rxn. were given to us.

Mini-prepped cultures ssb1121 #1-4 and ssb1134 #1-4 following Miniprep Purification of DNA Protocol

Suhani Vora 14:48, 8 March 2011 (EST)

Mapping Plasmid Preps

Followed Template Mapping Protocol for ssb121 #1-4 and ssb1134 #1-4

Ran Analytical Gels

Loaded Gel Lanes (Gel #1):
1. 5 ul Ladder

10. sbb1121 #1
11. sbb1121 #2
12. sbb1121 #3
13. sbb1121 #4


All look good: 3131 bp + 1031 bp

Loaded Gel Lanes (Gel #2):
1. 5 ul Ladder
2. sbb1134 #1
3. sbb1134 #2
4. sbb1134 #3
5. sbb1134 #4


All look good : 3131 bp + 525 bp

Suhani Vora 13:08, 10 March 2011 (EST)

Sent sbb1121 #3 and #4 for sequencing Sent sbb1134 #1 and #2 for sequencing

Both were correct.