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Team:Tec-Monterrey/ITESM14 notebook data.html - 2014.igem.org
Results: The Ap spread outward but stayed confined meaning the crosslinking worked. Adding the BCIP solution to the tube with the paper produced a noticeable discoloration. Adding the enzyme to the BCIP only produced a small discoloration. The binding of the alkaline phosphatase and BCIP to the paper was successful. The reaction worked only in the intended areas meaning that if the AP is bound to the factor c it may produce a signal only if it is cut, the AP nor BCIP disperses in the solution after being bound to the paper.
Objective:Before the fusion step, the hCG needs to be placed into the PSB1C3 plasmid, so that the hCG can be submitted. In order to add the hCG to add to the plasmid restriction sites need to be added this was done by adding the prefix and suffix through PCR.
Materials:Qiagen Master mix (2x), Prefix Primer, Suffix Primer, hCG DNA (diluted to 10ng/ul), and Nuclease free H20
Transformation of hCG-PSB1C3 ligation and overnight culture
Friday, August 11th.
Tori Radcliff
Colony PCR of hCG-PSB1C3 colonies
Saturday, August 12th.
Sunday, August 13th.
Tori Radcliff
Overnight culture of hCG-PSB1C3 colonies
Monday, August 14th.
Tori Radcliff
Miniprep of hCG-PSB1C3 cultures
Diagnostic restriction digest
PCR of factor-C gBlocks
Objective: Factor C was divided into 4 G-blocks and ordered from IDT. A PCR was performed to add overlaps to each block so that subsequent overlap extension PCRs could be performed.
A simulated gel was created and then the actual sampled of each were run on an e-gel. 5ul of product with 1ul of 6X dye and 14 ul of DI water was added to each well.
The wells include the following from left to right: Gene Ruler 1kb DNA ladder, PCR of g-block 1, PCR of g-block 2, PCR of g-block 3, PCR of g-block 4.
Wells contain the following:
Gene Ruler 1kb plus MW ladder, Reaction of blocks 1 and 2, Negative control (blocks 1 and 2), Reaction of blocks 3 and 4, Negative control (blocks 3 and 4), Reaction of 1 and 2 with 3 and 4 products, negative control (reactions 1 and 2 with 3 and 3 and 4), and Gene Ruler 1kb plus MW ladder.
Wednesday, August 16th.
Thursday, August 17th.
Tori Radcliff
Objective:Prepare hCG for ligation into pGEX through PCR
Materials:hCG DNA, primers forward and reverse and master mix
Equipment:Thermocycler
Procedure:Two 50 ul PCR reactions were performed. 25ul of qiagen mastermix, 2ul of block 5 DNA, 2.5ul of forward primer, 2.5 ul of reverse primer and 18 ul of nuclease-free water was added to tube 1 and tube 2.
Materials: digested vector cleaned by Cara Jones, T4 ligase and buffer, digested hCG
Procedure: Using a ligation calculator the concentration needed was 31.87 ng/ul in a 20ul total ligation. 1ul of PGEX at 105 ng/ul and 6 ul of hCG, 2 ul of T4 buffer, 1 ul of T4 ligase and 10 ul of nuclease-free water. A negative control was used which included digested mamba in pgex with no ligation buffer. The ligation reaction sat at room temperature for 10 minutes.
Transformation
Objective:To transform the ligation of hCG and PSB1C3
Results:There was no growth on the ligation plate nor negative control but there was growth on the positive control. Since there was no growth on the ligation plates that would mean the cells have no resistance to ampicillin and the ligation was inefficient due to the fact the hCG was not purified to eliminate contaminants ike EDTA and salts.