SBB10Ntbk-AmyKristofferson

BioE140L 2010 Homepage
My Project Page ==3/1/10: New plan of action Redo SOEing PCR, using a different temperature? 2K45? Use DMSO? (See PCR in Practice)

2/24/10: SOEing PCR and Analytical Gel

Wetlab Notes

• PCR gh1000F/ak11R on A+B+C using this protocol and 55 PCR.
• Ran analytical gel to verify that desired PCR product was created. (Protocol: add 3uL PCR product and 7uL of loading buffer)

My PCR product was labeled sbb22 and was in the fourth lane from the left. The PCR product should have been 619bp, but all the bands that showed up were under 500 bps.

• Performed a Zymo clean-up to remove dNTPs, Expand Polymerase, and oligos. What's left: the <500bp PCR product, A, B, C. DNA labeled "sbb22, final PCR product, AK" and placed in Box A.

Protocol Update

Next few steps:

Today:

Next time:

• Eco/Bam Digest
• Zymo clean up to remove small fragments and restriction enzymes
• Run a preparative gel to separate digested product from A, B, and C and to ensure product was correctly digested (Protocol: add 6uL PCR and 4uL of loading buffer)
• Cut out digested product and perform a Zymo clean-up

2/22/10: Preparative Gel for PCR product A,B,C

Mixed 6uL of each PCR product with 4uL of loading buffer and ran the gel.

My PCR products A, B, and C were in lanes 2, 3, and 4.

I cut out the bottom bands (the upper band is most likely template DNA), performed a small fragment Zymo Cleanup, and stored the eluted DNA in Box A.

2/17/10: New construction file, SOEing Protocol, PCR of A,B,C off of template

New sbb22 Construction File

Construction of FokI- basic part	
 PCR gh1000F/gh1001R on BBa_K243001   (261 bp, gp = A)
 PCR gh1001F/gh1003R on BBa_K243001   (264 bp, gp = B)
 PCR gh1003F/ak11R     Registry part BBa_K243001            (141 bp, gp = C)
 ---------------------------------------------------
 PCR gh1000F/ak11R on A+B+C           (619bp, EcoRI/BamHI)
 Digest pBjk2741-Bca1144              (EcoRI/BamHI, 910+2170, L)
 Product is pBjk2741-sbb22   {<FokI->}
 ---------------------------------------------------
 gh1000F  Forward for part <FokI-!    ccagtGAATTCatgAGATCTCAGCTGGTTaaatctgaactggaggag
 gh1001F  Making internal mutation 1+2 cgttggttccccgatcgattatggcgttatcgtggAcacaaaagc
 gh1001R  Making internal mutation 1+2 cgccataatcgatcggggaaccaacggtataaatggcaccGTctggtttac
 gh1003F  Making internal mutation 3   ccatatcaccaatTgcaatggggcagtgctgag
 gh1003R  Making internal mutation 3   ccattgcAattggtgatatgg
 ak11R    Reverse BamHI for FokI- CTGATggatccaaaattgatctcgccattg

SOEing Protocol for sbb22

• Set up PCR reactions according to your construction file as normal 33uL reactions as described in Cloning by PCR
• For each PCR, load 6uL of PCR product premixed with 4uL of loading buffer in a single well of a 1% agarose gel
• Cut out the bands, put them into a single 1.5mL microcentrifuge tube
• Add 650uL of ADB Buffer
• Proceed with the Zymo Gel Purification procedure
• Elute the DNA in 50uL of water
• Set up your second round of PCR as a normal 33uL reaction using the eluted mixture of fragments as template

Wet Lab notes

Prepared oligos and set up three PCR tubes using this protocol.

2/8/10: Construction Files for my parts

sbb22

Construction of FokI- basic part	
 PCR ak10F/ak20R     Registry part BBa_K243001            (230 bp, gp = A)
 PCR ak20F/ak30R     Registry part BBa_K243001            (71 bp, gp = B)
 PCR ak30F/ak40R     Registry part BBa_K243001            (245 bp, gp = C)
 PCR ak40F/ak11R     Registry part BBa_K243001            (135 bp, gp = D)
 ---------------------------------------------------
 PCR ak10F/ak11R on A+B+C+D           (619bp, EcoRI/BamHI)
 Digest pBjk2741-Bca1144              (EcoRI/BamHI, 910+2170, L)
 Product is pBjk2741-Bak1234  {<FokI->}
 ---------------------------------------------------
 ak10F  Forward EcoRI for FokI-      CCATAgaattcCAGagatctCAGCTGGTTaaatctgaactggaggagaa 
 ak20F  Base substitution 1  gtaaaccagACggtgccatt
 ak20R  Base substitution 1  aatggcaccGTctggtttac
 ak30F  Base substitution 2  cgttatcgtggAcacaaaagcg
 ak30R	Base substitution 2  cgcttttgtgTccacgataacgc
 ak40F  Base substitution 3  caccaatTgcaatggggcag
 ak40R  Base substitution 3  ctgccccattgcAattggtg
 ak11R Reverse BamHI for FokI-  CTGATggatccaaaattgatctcgccattg

sbb36

Construction of ColE2 Basic Part 
 Digest pBca9145-jtk2642                          (EcoRI/BamHI, 2057+485, S)
 Sub into pBca9523- Bca1144                        (EcoRI/BamHI, 2472+918, L)
 Product is pBca9523-jtk2642  {ColE2 ori med}