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 Analytical Gel.
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, Analytical Gel 1, 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