SBB10Ntbk-AmyKristofferson

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

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

2/17/10: New construction file, the protocol I'm following, set up PCR of A,B,C

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}