SBB11Ntbk-Amy Li

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AMY Li 14:06, 10 March 2011 (EST)

Today, I sent in two of each of my three products (six products total) for sequencing.

  • All 50ul of each purified ligation product was used
  • Primers flank the gene and adhere onto the backbone

AMY Li 15:50, 8 March 2011 (EST)

A 1kb ladder was used, and the three parts have digestion sizes: (1) sbb1125 {P_spy} at 3744=613+3131bp, (2) sbb1133 {P_ycgE} at 3484=353+3131, and (3) sbb1138 {P_yciW} at 4619=1488+3131. Arrows denote which products were sequenced

My colonies were picked over the weekend.

  • Followed protocol for picking colonies: Picking of colonies
  • sbb1125 contained of all white colonies, sbb1133 contained of mostly (~90%) pink colonies, and sbb1138 contained of few (~5%) pink colonies.
  • Picked four colonies for each of my three plates

Today, I mini-prepped my colonies.

I also digested my mini-preps.

  • I did this to preliminarily check if my part sizes were correct before I send two of each part out for sequencing
  • I threw out one darker-colored culture (remnants of a red bacteria suspected)
  • This yields 11 mini-prep digestions and one ladder, filling up 12 wells in a gel

AMY Li 13:26, 3 March 2011 (EST)

Today, I ligated my three inserts with their corresponding vectors.

Then, I transformed E. coli cells with the plasmid product from ligation.

  • Followed protocol for Transformation by heat-shock: Transformation by heat-shock
  • Letting shake in the 37 degree incubator for 45 minutes is sufficient (30 minutes bare minimum)
  • Plated 100 ul of cell-plasmid cocktail on kanamycin antibiotics plate.

AMY Li 15:31, 1 March 2011 (EST)

The three bands correspond to parts: (1) sbb1125 {P_spy} at 629bp, (2) sbb1133 {P_ycgE} at 369 bp, and (3) sbb1138 {P_yciW} at 1504bp. The smear of sbb1133 resulted from low DNA concentration.

Today, I redid my digestion because my digestion products mysteriously disappeared.

  • Used 2 ul of 10x Dye (ran 10ul of stuff)
  • Proceed to transformation with all three parts; though part sbb1133 (300+ bp) has a smeared band because of low concentration and may not work

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

A 1kb ladder was used. The three bands correspond to parts: (1) sbb1125 {P_spy} at 629bp, (2) sbb1133 {P_ycgE} at 369 bp, and (3) sbb1138 {P_yciW} at 1504bp

Today, I completed the digestion of my three PCR parts

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)

The three bands correspond to parts: (1) sbb1125 {P_spy} at 629bp, (2) sbb1133 {P_ycgE} at 369 bp, and (3) sbb1138 {P_yciW} at 1504bp

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
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 of Analytical Gel: 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}

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}

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}

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

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

Hello World!