SBB11Ntbk-VinidhraMani

Vinidhra Mani 13:57, 24 February 2011 (EST)

Today, we did the Zymo gel purification for the PCR products (sbb1111, sbb1135) this time using the CORRECT A4 BUFFER :) When we re-eluted I accidentally added a little too much water into sbb1111 and the volume is about 11ul instead of 8ul.

I also began redoing the Eco/Bam cut and paste for jtk2791 (lacZ gene). After the digest was prepared (37C for 1hr in the thermocycler following the protocol) a preparative gel was run, the larger band of interest was cut out and melted in ADB buffer at 55C. This was then placed in the freezer overnight for the next class period.

Vinidhra Mani 13:54, 22 February 2011 (EST)

We ran into some difficulties over the week. All of the buffer that we assumed was A4 for the zymo cleanups were actually AW buffer, containing quanidinium chloride which could easily mess with the DNA. Noticing this, Chris ran the PCRs and zymos again over the weekend and we were on track for the PCR digests done today. I did the digests for sbb1111 and sbb1135 (construction files listed earlier), using the digest protocol (8ul product, 1ul NEB buffer, 0.5ul EcoR1, 0.5ul BamHI). These were incubated at 37C in the thermocycler for 1 hour and then run on a preparative gel. The larger piece on both of these was cut out and melted in 600ul ADB buffer at 55C.

The Eco/Bam transfer for the lacZ gene needs to be run again as well, but the samples need to be prepared first.

Vinidhra Mani 14:28, 18 February 2011 (EST)

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
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