SBB10Ntbk-AmyKristofferson: Difference between revisions
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===SOEing Protocol for sbb22=== | ===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=== | ===Wet Lab notes=== | ||
Prepared oligos and set up three PCR tubes using [http://openwetware.org/wiki/Template:SBB-Protocols_PCR2 this protocol]. | Prepared oligos and set up three PCR tubes using [http://openwetware.org/wiki/Template:SBB-Protocols_PCR2 this protocol]. |
Revision as of 15:38, 22 February 2010
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 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}