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8/1/14
The PCR reaction to amplify the TdT G-block with the new forward primer was repeated with multiple changes. The annealing temperature was decreased from 56°C to 55°C. The contents of yesterday's PCR reaction was reproduced twice except the amount of Taq was doubled from 0.25μL to 0.5μL. Two other reactions were ran with double the amount of Taq as well as double the amount of TdT G-block (100pg instead of 50pg). One other reaction was ran was double the Taq, TdT G-block as well as 1μL of DMSO.After 40 cycles, the PCR products were ran on a 1.0% gel.
The gel showed that the amplification of G-block failed for all PCR reactions tested. Only the primers were amplified except for the reaction that included DMSO.
8/4/14
We single-digested 500ng of pET28b+_Tdt midiprep sample on 7/16 (1A, 43.5ng/μL) with NdeI and used 1μL of that sample to do PCR reactions.First PCR reaction was done with 1st truncated forward and regular reverse primer. In addition to digested (linearized) pET28b+_Tdt, circular DNA was also used. PCR was done with 95°C 30sec, 56°C 30sec, and 72°C 90sec (35 cycles). Then, another round of PCR was done with the same condition but 54°C annealing temperature. 1μL samples from each tube was taken from the first PCR and amplified again with new fwd (δEcoRI), 2nd, 3rd, 4th truncated forward primer. And left overnight. We will run gels tomorrow (along with the first product of linearized DNA, to make sure that first round of PCR went okay.)
8/5/14
We ran the gels to see if PCR went well, and concluded that 2nd round of PCR had so much background residues. 1st round of PCR went okay, so gel extraction was performed.8/6/14
In the morning STEM presentation was done and enzyme activities were done along with presentation.We set up another set of PCR with the same protocol from yesterday, but used gel extracted sample from the 1st round of PCR and increased annealing temperature to 55°C. Along with those samples, 1st round of PCR was repeated with single digested pET28b+_Tdt.
Left to Right: ladder | circular | linear | 1 | 2 | 3 | 4 | neg.control | ladder
8/7/14
Today we did a protein purification and extraction according to Bugbuster kit (Novagen) and Ni-NTA spin kit. We collected the lysate as well as the flow-throughs at step 5,6,7,8.We loaded the gel, but because the power supply did not work we could not run the gel and lost the lysate sample.
We also gel purified the bands from the gel that was run yesterday.
8/8/14
Today we were able to fix the power supply and run the gel on a polyacrylamide gel without the lysate sample. We then did a Coomassie stain on the gel to highlight the proteins. Because the step 5 flow through had so many proteins, it was unable to stay in its lane and expanded into the elution flow through lane, so the results of this gel was inconclusive. The gel is shown below:
Left to right: Ladder | Commercial Tdt | Flowthrough 5 with insert | 6 with insert | 7 with insert | 8 with insert | flowthrough 5 w/o insert | 6 w/o insert | 7 w/o insert | 8 w/o insert | Ladder
8/11/14
Today we inoculated pET28b+_Tdt in Rosetta cells from the glycerol stock and used pET28b+ in Rosetta cells transformation and put in the shaker overnight (37°C, 250rpm).Tdt experiments were done again, one with the protocol we used before and the other one with the protocol that company suggested.
8/12/14
Glycerol stock for pET28b+ in Rosetta cell is made and stored.4hr incubation in 20mL LB broth was done: samples were Tdt/IPTG treated, Tdt/neg, without insert/IPTG treated, without insert/neg.
With the gel extraction samples yesterday, double digestion was done with XbaI and PstI. Backbones (pSB1C3 and pSB1K3) were also digested, 50ng of vector and 75ng of insert were ligated and stored overnight at 16°C.
Gel electrophoresis with Tdt experiment was done with TBE/Urea precast gels. 10μL of samples were boiled with 10μL of TBE/Urea buffer for 10minutes, and was run with 200V, 40 mins. Gel was kept loaded without running for about 25 minutes because of the power supply problem.
8/13/14
We continued protein purification experiment, and lysed the cell with Bugbuster mastermix. Lysates were then purified with Ni-NTA spin kit and gel electrophoresis with protein precast gels were done. Buffers for Ni-NTA spin kit were re-made with new urea.8/14/14
Attaching secondary antibody was done in the morning according to Thermo Scientific/Bio-Rad protocols. PBST solution was made, stored, and used for wash steps.Transformation samples (pSB1C3_TdT and pSB1K3_TdT) were checked and pSB1C3 seems working. Will do colony PCR on monday, parafilmed and stored in the fridge.
8/15/14
We ran the gel electrophoresis with TBE/Urea Precast gels with TdT CleanAmp Experiment Samples.
Ladder | dATP | dATP 95°C | dCTP | dCTP 95°C | dGTP | dGTP 95°C | no TdT(neg) | 5min | 60min | Ladder
8/18/14
Colony PCR was done based on the transformation last week (pSB1C3_TdT and pSB1K3_TdT). Like last time, 2.5μL of samples were put into PCR reactions, and the other 2.5μL samples were applied to the plates. We will run gels tomorrow.8/19/14
Colony PCR samples were run with 0.8% gel. We did see bands with TdT sizes, but there were too much background noises that we cannot send for sequencing. Also, transformation samples were re-streaked like colony PCR, since the colony PCR done yesterday turned out to be RFP. If the plates just have red colonies tomorrow, we have to re-ligate and transform the samples with phosphotase treatments.Another set of TdT experiments were done with the variation of time. CleanAmp dATP and regular dATPs are incubated for 5, 10, 30, and 60 minutes. Samples are stored and we will run them when the gels are ready.
8/20/14
Checked the plates, and saw several white colonies. Should run PCR tomorrow since the other team was using the machine.Redid the double digestion of pSB1K3 and pSB1C3 for biobrick. Used Gel purified and PCRed TdT (9.4ng/μL) and double digested with XbaI and PstI. This time backbones are phosphotase treated, to make sure we don't get religated colonies.
8/21/14
Set up another colony PCR reaction, with the sample protocol last week. Used one colony from pSB1C3 plate, and 9 from pSB1K3 plates (plates were marked and stored in the fridge).We also ran a gel with TdT experiment samples,
Left to Right: Ladder | CleanAmp 5min | CleanAmp 10min | CleanAmp 30min | CleanAmp 60min | Regular dATP 5min | Regular dATP 10min | Regular dATP 30min | Regular dATP 60min
8/25/14
Gel purified PCR samples were extracted with QIAquick Gel Extraction Kit. Gene was eluted in EB buffer and pSB1C3_TdT, pSB1K3_TdT 3, pSB1K3_TdT 6, pSB1K3_TdT 7 was extracted, using new yellow spin column. Concentrations were written on the tube, and the samples were sent out for sequencing. 40ng of DNA in 10μL ddH2O and 5μL of 5μM primer (VF2 and VR for each sample) was mixed and ordered.Transformation of phospotase treated ligation samples of pSB1C3_TdT and pSB1K3_TdT in JM109 cells was done. Because everything was same except for phosphotase treatment, no controls were made and kept overnight in the incubator. Also, colonies from colony PCR were inoculated (pSB1C3_TdT 1, pSB1K3_TdT 1-9) and incubated overnight.
8/26/14
We re-inoculated minicultures from yesterday, but this time only the ones that we sent out for sequencing was re-inoculated (pSB1C3_TdT, pSB1K3_TdT 3, 6, 7) and kept overnight. (37°C, 300rpm). Glycerol stocks were also made with all minicultures and stored in -80°C.8/27/14
Midipreps for pSB1C3_TdT and pSB1K3_TdT 3, 6, 7 were done according to QIAGEN protocol. Airdried for overnight.8/28/14
Airdried samples were resuspended in 20μL TE buffer. should crank up the concentration.In order to test TdT's degradation rates in 95°C, the TdT Denaturation Experiment protocol was followed. TdT was subjugated to 95°C for different amounts of time and then allowed to react with a 24-mer oligo for 10 minutes. The gel shows that within 30 seconds in 95°C conditions, the enzymatic reaction rates significantly decreases. After 10 minutes, all TdT active is lost.
