SBB11Ntbk-Averee Chang

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Averee Chang 12:59, 10 March 2011 (EST)
Looked at the gel. I will be picking P_narP 2, P_narP, P_sfmC 1, P_sfmC 4 for sequencing after viewing.

Averee Chang 15:26, 8 March 2011 (EST)
Ligated and transformed the cut-and-paste part jtk2914 using Ligation of EcoRI/BamHI digestts and  Transformation by Heat Shock. I plated this part on a Spec plate and I will miniprep this part on Thursday. Today, I also mapped my parts, P_narP and P_sfmc using a mapping protocol using analytical digests. P_narP should be 620 bp while P_sfmc should be 723 bp.

Averee Chang 14:48, 3 March 2011 (EST)
I miniprepped the two parts I ligated and transformed. I occulated 4 colonies from P-narP and 4 colonies from P-sfmC. While I was re-suspending the bacteria, I noticed that one of the tubes from P-narP contained the parent vector (the cloned colony was red). So by the end of the mini-prep, I cleaned 7 tubes: 3 tubes of the P_narP part and 4 tubes of the P-sfmc part. For the mini-prep procedure, I used Miniprep Purification of DNA On Tuesday I will be ligating and transforming my cut-and-paste part, jtk2914.

Averee Chang 13:24, 1 March 2011 (EST)
Started the ligation of P_sfmC and P-narP. For both stress promoter parts, I used vector pBjh1601KC and will transform the part onto a lefty cell. For this procedure, I used the Ligation of EcoRI/BamHI digests protocol. I finished setting up the reaction at 10:18 and will continue with transforming the cells at 10:48am. Performed a Transformation by Heat Shock. Instead of adding 100μL of 2YT, I added 100μL of LB. I used the Green Stripe Plates, that contains KanR because vector pBjh1601KC contains the antibiotic sequence KanR.

Averee Chang 13:40, 24 February 2011 (EST)
On Tuesday, I learned that all my cleaned Cut-and-Paste and cleaned PCR products of P_sfmC and P_narP was contaminated because I used the wrong wash during the Zymo clean-up (I used the AW wash when I was supposed to use the A4 wash). So I had to start over with digesting and cleaning. Starting with my two PCR products, I did a gel prep, cut out the appropriate bands, and melted the gel with 600μL of ADB buffer. Today, I continued with a Zymo gel purification for my two PCR products. Now both parts, P_sfmC and P_narP have been digested and appropriately cleaned. I did a gel prep for my "cut-and-paste," cut out the bigger band, and cleaned with a zymo gel purification. By the end of today, both my PCR products and "cut-and-paste" parts have been digested and clean and will be ready for ligation and transformation on Tuesday.

Averee Chang 15:56, 18 February 2011 (EST)
I gel purified the part I digested with EcoRI and BamHI, jtk2914, using the protocol Zymo Gel Purification. My PCR product came back today (the one I re-did yesterday to make parts P-narP and P-sfmC) and I am currently running an analytical gel to ensure that my part got successfully amplified. To prepare for the analytical gel, I added 2.5 μL of blue dye and 6μL of the part for each part. When the gel picture came back,, my PCRs seemed successful. My two columns, column 9 and column 10 which are the last two on the far right, have bands at around 650 and 850 bp. This correlates with my part's length, 620bp for P_sfmC and 723bp for P-narP

Averee Chang 13:03, 3 February 2011 (EST)
Tuesday (February 15): Did a PCR to make P_narP from oligos ss34F, ss34R and P_sfmC from oligos ss52F,ss52R. Today, my PCR yield had too little volume (probably due to a loose cap on the PCR tube) and I re-did the PCR for both of them today using protocol Cloning by PCR Today, along with re-doing the two PCRs, I digested part jtk2914 from pBjh1601KC-jtk2914 using EcoRI and BamHI. After doing a preparation gel, I cut out the larger band of the two and melted the agarose gel and froze it in box "C." I digested with EcoRI/BamHI and underwent the preparation gel using EcoRI/BamHI Digest of PCR Products