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Team:NRP-UEA-Norwich/Week7
From 2012.igem.org
Welcome to the NRP UEA iGEM 2012 Wiki Lab Book
Please choose the relevant link to access our diary of that week!
Week Zero | Week One | Week Two | Week Three | Week Four | Week Five | Week Six | Week Seven | Week Eight | Week Nine | Week Ten | Week Eleven | Lab Protocols | Experiments
Contents |
. Research into sequencing companies to have our BioBricks sequenced.
. Finalising ordering of genes to be synthesised.
. Transformation of CFP, RFP and GFP by Rebecca.
. Plan of requirements for sequencing of B-M and M-B
. Message from GenScript confirming synthesising of ordered genes.
' Plates were found to not be contaminated but there was no growth on any of the plates.
. Retransformed CFP, RFP and GFP into alpha competent cells and plated. Positive controls were made. Some of the transformation culture was retained and more LB medium was added and grown overnight.
. Planning of growth study. Growth Study protocol is on the Lab Protocol page
. The transformation was successful and therefore, inoculations of CFP, RFP and GFP were made.
. Redid the growth study using Alpha cells, PyeaR transformed cells, B-M transformed cells and M-B transformed cells. The experiment lasted 12 hours to give a representative view.
. Restriction digest of B-M and M-B using Pst1 and Spe1.
. Minipreps of M-B and B-M.
. Minipreps of CFP, RFP and GFP.
. Ran the restriction digest on a gel.
. Ran a lag phase only growth study which involved starting the cultures at a lower absorbance and hence concentration.
. Arrival of primers.
. iGEM PCR primer stock made up to 100µM, then further diluted.
. Preparation of plasmid DNA of our BioBricks to be sent for sequencing. 6µL of the DNA was placed into separate eppendorfs to be sent off.
. Rachel carried out a gel purification (on both MB and BM from the gel run) using promega wizzard kit (Ref: A9281) as seen in the lab protocol page. From the gel, the lanes containing BM1, BM2, BM3, BM4, MB1, MB2 and MB4. These refer to bacterial mammalian and the mammalian bacterial promoter transformed cells from different colonies. The purified samples were stored in the freezer.
. Rebecca transformed BioBrick BBa_K561002 (PFDHF +RFP + TETR) into Bioline Alpha Select Gold Standard cells following the transformation protocol as seen in the lab protocols page. The negative control was simply alpha cells, untransformed. The transformation was plated onto agar plates containing chloramphenicol resistance at 25µg/ml.
. Khadija nanodropped the miniprepped plasmid BioBricks BBa_K206009 (GFP), BBa_E0420 (CFP) and BBa_K081014 (RFP). As many cultures were grown per a single colony on the plates, there were separate epppendorfs containing the plasmid DNA. In total there were 4 of CFP, RFP and GFP samples. These were shown to have high concentrations of DNA ranging from 165ng/µL to 332 ng/µL. Following the nanodrop a restriction digest was carried out to validate the plasmid DNA. Two sets of restriction digests were carried out. One is to cut at Pst1 of the flanking suffix/prefix DNA and the other digest is to cut a specific restriction site in the backbone. As BBa_K206009 is found in pSB1AK3 there is a 'Hind'III site and BBa_K081014 is in pSB1C3 and contains an EcoRV site. BBa_E0420 contains many restriction sites but none that can be cut by enzymes that we have in immediate stock. Therefore, a second restriction digest was not carried out on this BioBrick. The single digests involved 1µL DNA, 2µL buffer, 0.2µL BSA, 0.5µL of restriction enzyme and 16.3 µL of distilled water. Buffer H was used in the digests using EcoR1, Buffer B for HindIII and Buffer D for EcoRV. These were stored in the freezer after 2 and a half hours of digestion.
