De Novo Synthesis Lab Notebook
6/3/14
Transformed DH5alpha with pET28b (+) vector on two plates, a Kan and an Amp. They were then left to grow overnight.
6/4/14
The plates were checked first thing in the morning and no colonies were found. Therefore we went through the procedure of transforming DH5alpha with new C media and two different dilutions of the pET28b (+) vector as well as two control strains(pET26 (+) in DH5alpha, pEt28 + CDS7) that were known to grow on Kan plates. They were then left to grow overnight. This time we did not use the vortex for mixing and were much more gentle with the cells.
We also transformed DH5alpha with 6 different BioBrick plasmids (pSB1A3, pSB1C3, pSB1K3, pSB1T3, pSB2K3), two BioBrick RBS (B0030, B0034), and a BioBrick double terminator (B0015). They were then left to grown overnight.
Three 1% agarose gels were then prepared, with one containing about 100mL of mixture and the other two containing about 50mL of mixture.
Tomorrow, we need to check for cell growth on all plates and further grow any colonies that have formed.
6/5/14
First thing in the morning, we checked all plates and found that colonies had grown on all of them. Colonies from three of the plates (DH5alpha, DH5alpha and pET28b+, and DH5alpha and pSB1C3) were extracted and allowed to incubate overnight. Parafilm was used to secure the other plates so that they can be incubated at a later time.
6/10/14
Yesterday, Wilfrido and Joe took the three minipreps of pET28b and three minipreps of pSB1C3 and cut them using the enzymes Xho1 and Xba1 respectively to create three new samples each.
Today, we created a 0.8% agarose gel for the pET28b samples and a 1.5% agarose gel for the pSB1C3 samples. Once prepared, the gels were run. The gels could be read from left to right as: ladder, uncut 1, uncut 2, uncut 3, cut 1, cut 2, cut 3. There was an extra band at the top of all of the samples which we suspect is due to the fact that we used 100x BSA instead of 10x BSA. All of the samples appeared normal except for the third uncut sample of pET28b, which was missing a band. As a result, we discarded this sample.
6/11/14
A couple of 1.0% Gels were made.
6/12/14
Today, we realized that the cells that we transformed with the pSB1C3 and pSB1T3 plasmids were not done properly due to the fact that they did not express the red protein. We attributed this fault to errors with the BioBrick plates. As a result, we retransformed DH5alpha with these same plasmids but from other wells and left them to incubate overnight. We also made six tetracycline plates.
6/13/14
The cultures of the pSB1C3 and pSB1T3 once again appear to not have been successful. The pSB1C3 grew many small colonies none of which expressed the red protein and the pSB1T3 did not grow any colonies. We believe that the wells have defective plasmids. The wells are 2014, plate 1, well 23O; 2011, plate 1, well 7A.
6/16/14
We took two cultures (pSB1A3 and pSB1K3) that showed colonies that expressed the red protein and are growing them overnight in LB broth with their respective antibiotics.
Double restriction digested pSB1C3 and pSB1T3 which are both linearized with enzymes EcoR1 and Pst1. The concentrations are 10ng/ul. The total volume for each was 20 microliters.
6/17/14
The cells grown overnight were checked and were confirmed to have the correct plasmids, pSB1A3 and pSB1K3, due to the red color of the LB solution. A 1mL miniculture of equal part cells and a 60% glycerol stock were prepared for each sample and stored in the -80C freezer.
The rest of the overnight cultures, three samples of DH5alpha with pSB1A3 and three samples of DH5alpha with pSB1K3, were minipreped following the Miniprep Protocol with some exceptions. The 5mL of overnight culture was centrifuged at 3900rpm for 10 minutes for the first step of preparing the cell lysate. For the second step of eluting the DNA, 50ul of preheated TE buffer was added to the column instead of the stated 75ul.
After the plasmids were isolated, the concentration was measured using the nanodrop. Samples 1, 2, and 3 of pSB1A3 was measured to be 142, 211, and 197 ng/ul, respectively and samples 1, 2, and 3 of pSB1K3 was measured to be 139, 161, and 152 ng/ul. The plasmids were stored at -20°C.
Double restriction digest reactions were prepared for each sample of pSB1A3 and pSB1K3 with the restriction enzymes EcoR1 and Pst1. An error was made in the procedure in which 100X BSA was added instead of a diluted 10X BSA. The solution concentrations are 15ng/ul with a total volume of 20 ul.
6/18/14
We ran the gel to verify that the isolated plasmids are pSB1A3 and pSB1K3 due to their base pair lengths and that the digestion was successful. We then ligated both plasmids with J04450. After we transformed the plasmids into DH5alpha and left it to grow on plates overnight.
6/18/14
The cells left to grow overnight grew colonies successfully except the red protein was not expressed. The plates were parafilmed and left in the fridge to allow additional time for the cells to produce the red protein. Due to the colonies having similar traits to the first colonies that were transformed with pSB1C3 and pSB1K3, large amount of colonies and small colony sizes that failed to express the red protein, it is suspected that the competent cells used were contaminated with another type of bacterial.
DH5alpha competent cells from new stocks were re-transformed with the plasmids as well as using a 100:1 LB/glucose solution in place of the 1X SOC and were left to grow overnight. Two controls were also prepared: cells from the old competent cell stock were prepared in the LB/glucose solution and grown on a chloramphenicol plate and cells from the new competent cell stock were prepared in the old SOC media grown on a chloramphenicol plate.
A double digestion reaction was prepared for pET28b+ with the restriction enzymes BamHI and NdeI. The cut plasmid was stored in the -20C freezer.
Three samples of DH5alpha colonies transformed with the pET28b+ plasmid that were previously grown on a plate were minicultured and left to grow overnight on the shaker.
6/20/14
Despite being in the fridge overnight, the colonies transformed with the ligated pSB1C3 and pSB1T3 on plates did not start expressing the red protein marker. This suggests that the colonies were in fact not DH5alpha but a bacteria resistant to various antibiotics.
Only one of the four plates incubated overnight grew colonies. The cells from the old competent cell stock prepared with a LB/glucose solution grew on a chloramphenicol plate while the cells from the new competent cell stock prepared in the old SOC media did not grow any colonies. The colonies that grew has similar colony sizes and numbers with the unknown strain of bacteria which suggests that the old competent cells are indeed contaminated.
The DH5alpha from the new competent cell stock without any plasmids, as expected, did not grow on the chloramphenicol plate. However, the cells transformed with pSB1C3 also did not grow despite being plated on a less concentrated antibiotic plated (50ug/ml instead of 150ug/ml). The cells transformed with pSB1T3 on a tetracycline did not work as well. The plan for the future is to retry growing cells transformed with the plasmids with new competent cells while also transforming cells with the plasmids in the transformation efficiency kit to test if the transformation protocol is correct.
Two more restriction digest reactions were prepared with 100ng of DNA each; a single digest of the pET28b+ with BamHI and another single digest with NdeI. Two 0.8% gels were ran, each with each sample of the plasmid with an uncut plasmid, double digested plasmid and the two single digested plasmid. The greater intensity of the double digested DNA bands are due to the fact that the concentration of the double digested DNA is several folds greater than that of the uncut and single digested DNA. As seen from the ladder, the plasmid is a bit greater than 5.3kbp which matches the known length of pET28b+, confirming the identity of the plasmid. The similar lengths of the single digested plasmid suggest that both enzymes are probably working however, from the large amount of uncut DNA in the double digested DNA lane, the enzymes are probably as active as it should be. For future digestions with BamHI and NdeI, fewer DNA will be used as well as longer incubation times to assure the digestion runs to a completion.
After the confirmation of the plasmid, the three minicultures prepared were minipreped. The isolated pET28b+ were stored in the -20C freezer.
6/23/14
Prepared Ampicillin plates and two 0.8% gels. Additionally a 6% gel was made in preparation for the CleanAmp TdT experiment which will be run tomorrow. We were going to run the experiment today but it still needs to be tweaked in order to get usable and distinguishable results on the gel.
(JWP: double digested pET28b+ with Nde1 and BamH1-HF for 3hrs, and stored in the freezer. Will run gel with other samples tomorrow)
6/24/14
Today we performed four experiments using the commercially purchased TdT, dNTPs, and Clean Amp heat labile dNTPs. The protocol "Clean Amp Experiment Protocol" was followed for two of these experiments while the other two were used as control. For the positive control, TdT was mixed with the dNTPs in TdT buffer along with CoCl2 and ddH2O and allowed to incubate in a 37 C water bath for 30 minutes. The reaction was stopped by placing the mixture in a 70 C water bath for 10 minutes. For the negative control, the same procedure as for the positive control was followed without the addition of dNTPs. All of the experimental products were stored at -20 C. We plan on running the products through a 6% gel tomorrow morning so that it can run all day long.
The double digested pET28b vectors from yesterday were run on a 0.8% gel along with the original uncut pET28b vector and the single digested pET28b vectors.
The double digested pEt28b vectors that were cut out of the agarose gel on Friday were gel extracted and placed in the -20C freezer.
6/25/14
Had Gel electrophoresis of CleanAmp tdt experiment from 6/24, with 6% gel, 30V, for 5 hrs and 22 mins.
- As a ladder 0.34 uL of 33 Mer, 2.5 uL 52 Mer, 0.43 uL of 42 Mer, 0.59 uL of 24 Mer were added with 21.14 uL of ddH2O, and 4.2 uL of 6x dye was added.
- To see the results, stained with the solution of 500 uL of Ethidium Bromide(10ug/ml) in 400 mL water. 400 uL was added first with 400 mL of water and stained for 10 minutes, but 100 uL was added later and stained for another hour and fifteen minutes.
- Because of the low voltage, the shape of the wire was influencing the overall shape.
Built truncated tdt primers with the reference of 2004 Rephasky paper. The four sequences are uploaded to Google Drive under Tdt Info.
Miniprepped PSB1C3 (1&2) according to Invitrogen protocol with centrifugation and were stored in the box.
Additionally a 6% gel was made in preparation for additional cleanAmp tdt experiment
6/26/14
Checked the picture of the 6% gel from yesterday, and repeated the same CleanAmp Tdt experiment with newly arrived DNTPs. The procedures were the same, but we divided the experiment into two different DNTP's, one with A and one with G. This time we made sure to add Tdt to the controls. Ladders were also prepared same as last time, but dye has to be added before running the gel (which will be 4.2 uL).
6/30/14
Because of a mistake the gel was run in the opposite direction, this did not leave us with enough to re-run the gel so the CleanAmp TdT experiment was repeated exactly as it was done on 6/26. The plan is to run these results on the same gel first thing tomorrow morning. Additionally a preliminary outline of the procedure for the actual TdT experiment was made but still needs significant revisions and more research before it is feasible to do.
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