SBB11Ntbk-VinidhraMani
Vinidhra Mani 19:28, 20 February 2011 (EST)Headline text
Today, for the toxic gene digest, after the gel was fully melted in buffer and placed in the freezer, a zymo gel purification was done, following the procedure and the sample was then eluted back with 8.5ul water.
Vinidhra Mani 13:47, 17 February 2011 (EST)
2/17/11 Today, for the completed PCR reactions, the analytical gel was prepared and run. (parts sb1111 and sb1135 for P_oppBCF and P_phrB). 2ul of the PCR product and 5ul of loading dye were added to a 500ul tube, as preparation for analytical gel. The gel revealed desirable results, so the next step in this process will be conducted in the next full lab period.
In addition, the digest for the toxic gene (lacZ) was done. 8ul of the PCR product (pBca9145-jtk2791) was mixed with 1ul NEB buffer and digested with 0.5ul each of EcoR1 and BamHI. Subsequently, this was placed in the thermocycler at 37C for 1 hour. After the digest was completed, a preparative gel was run and the corresponding band of greater length 3110 was cut out. In the preparative gel stage, the concentration of DNA was very high and therefore needed to be run for a little extra time in order to be separated effectively. As a result, instead of 2 bands, 3 bands were seen (the extra band as a result of high DNA concentration relative to restriction enzyme, not cutting all of the DNA). After the band of interest was cut out, the gel piece was placed in ~600ul of ADB buffer and melted at 55C, then placed in the freezer as per protocol.
Vinidhra Mani 14:08, 15 February 2011 (EST)
2/15/11 Today, we did our first simple PCR reactions. I completed the reaction for part sbb1111 and sbb1135, the P_oppBCF and P_phrB promoters, respectively. The PCR protocol listed on our SBB homepage was used. Hopefully this will work the first time! I still have one part to make given that this works, that is the Eco/Bam transfer for the lacZ gene.
Construction files:
1) Part sbb1111 {P_oppBCF} PCR ss36f/ss36r on MG1655 gen. (1519bp, EcoRI/BamHI) Sub into pBjh1601KC-Bca1144#5 (EcoRI/BamHI, 3131+910, L) Product is pBjh1601KC-sbb1111 {P_oppBCF}
ss36fForward Cloning of P_oppBCF aaaccGAATTCatgAGATCTctcggcgacacgattctacaactttttgg ss36rReverse Cloning of P_oppBCF tttggGGATCCagactttcgcgtctttattatcccagg
2) Digest pBca9145-jtk2791 (EcoRI/BamHI, 3110+2057, L) Subclone into pBca1766-Bca1089 (EcoRI/BamHI, 3654+696, L) Product is pBca1766-jtk2791 {LacZ}
3) Part sbb1135 {P_phrB} PCR ss62f/ss62r on MG1655 gen. (1008bp, EcoRI/BamHI) Sub into pBjh1601KC-Bca1144#5 (EcoRI/BamHI, 3131+910, L) Product is pBjh1601KC-sbb1135 {P_phrB}
ss62fForward Cloning of P_phrB aaaccGAATTCatgAGATCTtaaagccttcgaggaaaaatttccgc ss62rReverse Cloning of P_phrB tttggGGATCCgcattgataagttcagcctg