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AMY Li 13:45, 24 February 2011 (EST)

Today, I completed the digestion of my three PCR parts
- Followed protocol for Zymo Gel Purification: Zymo Gel Purification
- Eluted PCR product in 8ul ddH2O

Next step: Ligation

AMY Li 13:30, 22 February 2011 (EST)

Today, I began the digestion of my three PCR products
- Performed the protocol for EcoRI/BamHI digestion of PCR Products: EcoRI/BamHI Digest of PCR Products
- Followed up with gel extraction with 1ul of 10x loading dye
- Stopped after melting gel in 600 ul ADB buffer

Next step: - Continue gel extraction/purification to complete digestion and get pure DNA

AMY Li 13:29, 17 February 2011 (EST)

This is the roadmap for my project:
For each of my three parts:
0) Amplify part using PCR
1) Analytical Gel to see if my PCR products were successful
2) Zymo Clean-up to isolate only DNA part; do not run another analytical gel
3) Digestion using EcorRI and BamHI
4) Ligation
5) Transformation

Today, I retrieved my three PCR products and ran them through analytical gel.
- Ran an analytical gel on my PCR products to visualize them
- Used 5ul Loading Dye and 2ul PCR product
- Not using Preparation Gel because don't need to gel extract

Results: PCR products were successful!
- Well 1: PCR Part sbb1125 {P_spy} 629 bp
- Well 2: PCR Part sbb1138 {P_yciW} 1504 bp
- Well 3: PCR Part sbb1133 {P_ycgE} 369 bp

Today I also column purified my PCR products by Zymo Clean-up
- Followed protocol for Regular Zymo Clean-up: Regular Zymo Cleanup
- For all "spin through"s, spun at 30 seconds at 14.5rpm (max RPM)
- Eluted each part with 33ul ddH20

Next Steps: Digestion with EcorRI/BamHI, Ligation, then Transformation.

AMY Li 17:04, 15 February 2011 (EST)

Today, I set up three cloning by PCR reactions, one for each of my three promoter parts.
- Followed the protocol for cloning DNA: Cloning by PCR
- Resuspended ALI_005 to 100uM and diluted it to 10uM
- Used the Expand buffer and polymerase for my PCR because the part templates were all from MG1655 gDNA
- PCR Thermocycler program 2K55 because all PCR products are less than 2kb

AMY Li 17:04, 15 February 2011 (EST)

Construction Files for my three Bgl Bricks basic parts for the random ball project:

Part sbb1125 {P_spy}
PCR ss51r/ss51f on MG1655 gen. (629bp, EcoRI/BamHI)
Sub into pBjh1601KC-Bca1144#5 (EcoRI/BamHI, 3131+910, L)
Product is pBjh1601KC-sbb1125 {P_spy}

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ss51f Reverse Cloning of P_spy tttggGGATCCcatgtcctgatgcggaccgaacttgcc
ss51r Forward Cloning of P_spy aaaccGAATTCatgAGATCTtggcgcaggacggagaggaacg

Part sbb1138 {P_yciW}
PCR ss65r/ss65f on MG1655 gen. (1504bp, EcoRI/BamHI)
Sub into pBjh1601KC-Bca1144#5 (EcoRI/BamHI, 3131+910, L)
Product is pBjh1601KC-sbb1138 {P_yciW}

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ss65f Reverse Cloning of P_yciW tttggGGATCCgtagaaagggatcgctgacc
ss65r Forward Cloning of P_yciW aaaccGAATTCatgAGATCTcggtgctggagaatattctgcagg

Part sbb1133 {P_ycgE}
PCR ss60f/ALI006 on E. coli MG1655 gDNA (369 bp, EcoRI/BamHI)
Sub into pBjh1601KC-Bca1144#5 (EcoRI/BamHI, 910+3131 bp, L)
Product is pBjh1601KC-sbb1133 {P_ycgE}

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ALI005 Forward cloning of P_ycgE CCATAgaattcatgagatctGTTTGCTAAAGCTAAATTGAATGGTATCC
ss60f Reverse cloning of P_ycgE tttggGGATCCggcgttgccaggcccggagagtgacag

AMY Li 23:04, 7 February 2011 (EST)

Hello World!