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)


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.
 * Followed protocol for mini-prepping DNA: Miniprep purification of DNA
 * Mini-prepped 12 culture tubes in total

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.
 * Followed protocol for Ligation of EcoRI/BamHI digests: Ligation of EcoRI/BamHI digests

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)


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)


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 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!