SBB09Ntbk-DaviddeRenzy

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David De Renzy 14:53, 18 February 2009 (EST)
AND
 * Reconstituted oligos to 100μM
 * Ran basic PCR cloning reaction
 * PCR Odd001F/Odd002R on E. Coli plasmid pAPEC-1 (AF218073)	(1603 bp, EcoRI/BamHI
 * Into PCR tube labeled M10040
 * Odd001F	Forward EcoRI oligo for TshA	ccataGAATTCatgAGATCTacacttttacctgtatacg
 * Odd002R	Reverse BamHI oligo for TshA	ctgatGGATCCtcagaatgaataacgaatattagcg
 * PCR Odd003F/BBa_G00101 on pBca1256-Bca1346		(3722 bp, EcoRI/BamHI)
 * Into PCR tube labeled M10041
 * Odd003F	Forward oligo for INP			ccaaaGAATTCatgAGATCTtgtaATGaatctcgacaaggcg
 * BBa_G00101 	Reverse sequencing of pSB1A* plasmids	attaccgcctttgagtgagc

Next step -> Run Analytical Gel

David De Renzy 15:38, 23 February 2009 (EST)

 * Ran Analytical gel on M10040 & M10041
 * 5μL PCR product + 5μL blue loading buffer (concentration was weak)
 * Both expected products were present
 * M10041 had a small fragment (~1000bp) of unknown DNA
 * Performed Zymo clean-up on M10040 & M10041
 * Eluted final products into 33μL water

Digest and gel purify M10040 & M10041 Maybe, run another analytical gel & purify with remaining "zymo-cleaned" M10041 OR Run a more stringent PCR for M10041 with higher annealing temperatures

David De Renzy 14:21, 25 February 2009 (EST)

 * Digested M10040 & M10041
 * Used 8μL of each PCR product for digest
 * Gel purified M10040 & M10041 Digests
 * Also gel purified 15μL of M10040 PCR product (after zymo clean-up)

Ligation Transformation

David De Renzy 14:58, 27 February 2009 (EST)

 * Performed ligation of EcoRI/BamHI digests for pBca9495KC-M10040 & pBca9495CA-M10041
 * Transformed ligation products into E. coli cells
 * Plated pBca9495CA-M10041 transformed cells onto a CA (ampicillin) selective plate
 * Plated pBca9495KC-M10040 transformed cells onto a KC (Kanamycin) selective plate

Next-Pick colonies

David De Renzy 14:14, 2 March 2009 (EST)

 * NO COLONIES FOUND!
 * Sources of Error:
 * No gel purification was performed after digestion
 * For pBca9495CA vector, proper extended incubation period at 37°C for 40 mins was not performed
 * Started back at digestion step
 * Used M10040 Product after Zymo cleanup
 * Used M10041 product after Zymo cleanup and gel purification (to remove 1kb unwanted fragment
 * Gel purification
 * column1=M10040, column2=M10041, column3=Ladder
 * Cut out gels were each place in 600μL of ADB buffer in labeled eppendorf tubes

Next-> finish gel purification Maybe start ligation/transformation

David De Renzy 14:24, 4 March 2009 (EST)

 * Zymo gel purification of M10040 & M10041 completed
 * eluted into 8.5μL of ddH20 for each
 * Performed ligation of EcoRI/BamHI digests for pBca9495KC-M10040 & pBca9495CA-M10041
 * Transformed ligation products into E. coli cells
 * Performed 40 min shake & incubation at 37°C with 100μL LB added for both products
 * Plated pBca9495CA-M10041 transformed cells onto a CA (ampicillin) selective plate
 * Plated pBca9495KC-M10040 transformed cells onto a KC (Kanamycin) selective plate
 * Note: Amp=Red, K=green, C=blue

Next-pick colonies

David De Renzy 13:13, 6 March 2009 (EST)

 * ~20 colonies on pBca9495KC-M10040 plate
 * 2 colonies were already picked and incubated overnight by the GSI's
 * Minipreped and eluted with 50μL of water
 * Loaded sequencing plates: Plate1=H3 (M10040 MP clone1) & Plate2=A2(M10040 MP clone2)
 * NO COLONIES pBca9495CA-M10041 plate!
 * This part will have to be abandoned

Next- Mapping, wait for sequencing results

David De Renzy 15:55, 9 March 2009 (EDT)

 * Digested and mapped M10040 clone 1 and clone 2