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Team:NRP-UEA-Norwich/Week8
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Contents |
. Khadija carried out a double digest on CFP, GFP and RFP. They were digested with Pst1 and Xba1 and left overnight. The mastermix was made using 10µl Biffer H, 1µl BSA, 2.5µl Xba1 and 2.5µl Pst1. This was evenly distributed into 5 tubes. The 5 tubes contained: 3µl GFP and 13.8µl of water (nuclease free), 3.94 µl GFP and 12.86 µl water, 3.47 µl RFP and 13.33 µl, 3.59 µl RFP and 13.21 µl water and 3.6 µl CFP and 13.2 µl of water.
. The single digests from the previous Friday were run on a 1% agarose gel. The gel showed that GFP was cut once and linearised, as expected, if cut by either Pst1 or EcoRV. The fragment length was estimated to be around 3000 bp. If the linearised fragment was GFP, it would be around 2800 bp. The digest of the RFP BioBrick produced two fragments. This should not have occurred. We decided to repeat the single digest of RFP. . The single digest of RFP was carried out alongside the single digest of CFP. We decided only to do single digests with Pst1 to validate. The quantities remained the same. 2µl Biffer H, 0.2µl BSA, 0.5µl, 1 µl DNA and 16.3 µl of water. These were left overnight to digest (both the single and double digests).
. Looking at the plates from Friday which had been stored in the fridge after a day in an incubator, we saw growth on the plates. Both the negative and positive controls showed the expected level of growth. We inoculated the plates containing colonies transformed with the BBa_K561002 (PFDHF +RFP + TETR) into media culture with chloramphenicol. These we left in a shaking incubator overnight at 37degrees. In total three colonies were inoculated into three 5 ml media tubes.
. Liaisons with BioSciences continues to buy sequencing vouchers.
. A 1% agarose gel was made by Rebecca and poured whilst Khadija began setting up the completed restriction digest products to be run on a gel. 15µl of the single digests were pipetted into 5µl of loading dye whilst 20µl of double digested DNA was mixed with 3µl of loading dye. Into the wells all the prepared samples were loaded. The electrophoresis took place for 1hour and 30mins.
. After running the gel, we noticed the bands were not straight. We then noticed the gel itself had not set straight therefore, with inconsistent thickness of the gel, we suspect this could have affected the outcome. We found that whilst the cut out fragments were what we expected them to be ~800bp, the plasmid backbone was evenly 1000bp out. We decided to repeat the digests.
. The single digest was completely redone. As originally multiple colonies were inoculated, we decided to digest all the samples we had. In total we digested 11 samples (3 GFP, 5RFP and 4 CFP). Again the quantities in each were as follows 2µl Buffer H, 0.2µl BSA, 0.5µl, 1 µl DNA and 16.3 µl of water. These were all run overnight.
. The grown overnight cultures of PFDHF + RFP + TETR were miniprepped. The concentrations of these have yet to be determined.
. A 1% agarose gel was made and the overnight single digests of CFP, RFP and GFP were run on the gel. 2 samples of each were run alongside the uncut versions. The gel confirmed GFP being the BioBrick we were after. The others again showed different lengths, therefore, we can confirm they were not what we predict them to be.
. Following this the GFP samples from which the digests were made from underwent a double digest using Xba1 and Pst1. The double digest is separate the GFP insert from the plasmid backbone so that we can isolate the DNA from gel and ligate it to our BioBrick: M-B and B-M. The GFP samples that we ran the doube digest on had the following concentrations: GFP1 = 332.9ng/µl, GFP2 = 253.7ng/µl, GFP3 = 255.8ng/µl and GFP4 = 267.1 ng/µl. Using these figures, 3µl of GFP1, 3.94µl of GFP2, 3.9µl of GFP3 and 3.79µl of GFP4 was pipetted into ependorfs with 3.2µl of a master mix solution consisting of 2.5µl of Buffer H, 1µl BSA, 2.5µl Xba1 and 2.5µl Pst1. Water was added to each of these to make them up to 20µl. This was left overnight.
. Following yesterday's minipreps of PFDHF + RFP + TETR, we nanodropped the isolated plasmids. The concentrations were very high. With 3 inoculations made from each colony, these repeats of each colony were labelled a, b and c. The concentrations were as follows:
1a) 656.3ng/µl 1b) 550.1ng/µl 1c) 502.2ng/µl
2a) 423.1 ng/µl 2b) 399.8ng/µl 2c) 347.7ng/µl
3a) 585.0ng/µl 3b) 440.1ng/µl 3c) 522.4ng/µl
Once again Lukas has proved himself as King of minipreps!
. As we are running low on the isolated plasmids of B-M and M-B, we decided to get more by carrying out a single digest on M-B and B-M. The single digest is to confirm their identity and then we will run a double digest where we will then isolate the fragment. The single digest involved using 4µl of DNA, 2µl Buffer H, 0.2µl BSA, 0.5µl Pst1 and 15.3µl water. The single digest was run for 3 hours. This was then stored in the freezer for the night.
