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Team:Tec-Monterrey/ITESM14 notebook data.html - 2014.igem.org
Monday, May 1st.
Sunday, June 1st.
Monday, June 2nd.
Tuesday, June 3rd.
Wednesday, June 5th.
Thursday, June 5th.
Friday, June 6th.
Saturday, June 7th.
Sunday, June 8th.
Monday, June 9th.
Tuesday, June 10th.
Wednesday, June 11th.
Thursday, June 12th.
Friday, June 13th.
Saturday, June 14th.
Sunday, June 15th.
Monday, June 16th.
Tuesday, June 17th.
Wednesday, June 18th.
Thursday, June 19th.
Friday, June 20th.
Saturday, June 21st.
Sunday, June 22nd.
Monday, June 23rd.
Tuesday, June 24th.
Wednesday, June 25nd.
Thursday, June 26th.
Friday, June 27th.
Saturday, June 28nd.
Sunday, June 29nd.
Monday, June 30nd.
Tuesday, July 1st.
Wednesday, July 2nd.
Thursday, July 3rd.
Friday, July 4th.
Wednesday, July 5th.
Thursday, July 6th.
Friday, July 7th.
Tori Radcliff
Objective:To determine if Alkaline Phosphotase will bind to nitrocellulose paper and produce a signal when mixed with BCIP.
Materials:Cut small strips of nitrocellulose paper (Nitran), Promega Alkaline Phosphatase (Conc: 1ng/ul), Alkaline 10X buffer diluted, BCIP
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.
Saturday, July 8th.
Sunday, July 9th.
Monday, July 10th.
Tuesday, July 11th.
Wednesday, July 12th.
Thursday, July 13th.
Friday, July 14th.
Saturday, July 15th.
Sunday, July 16th.
Monday, July 17th.
Tuesday, July 18th.
Wednesday, July 19th.
Thursday, July 20th.
Friday, July 21st.
Saturday, July 22nd.
Sunday, July 23rd.
Monday, July 24th.
Tuesday, July 25th.
Wednesday, July 26th.
Thursday, July 27th.
Friday, July 28th.
Saturday, July 29th.
Sunday, July 30th.
Monday, July 31st.
Tuesday,August 1st.
Tori Radcliff
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.
Tuesday, August 15th.
Tori Radcliff
Overlap Extension PCR
Objective:To combine the four PCR-ed g-blocks of factor-C.
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.
Tatenda Tela
Overnight cultures
Colony PCR of Cara's transformants
Friday, August 18th.
Saturday, August 19th.
Sunday, August 20th.
Monday, August 21st.
Tatenda Tela
Miniprep
Ligations
Tuesday, August 22nd.
Wednesday, August 23rd.
Thursday, August 24th.
Friday, August 25th.
Tori Radcliff
Objective:To repeat the double digest of PSCB1C3 and hCG PCR
Materials:DNA of Lt10-PCB1C3 (80 ng/ul) and hCG PCR product, Restriction enzymes: NotI, EcoRI, XbaI, PstI,SpeI, Green Fastdigest Buffer (10x), and Nuclease free H20
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.
Wednesday, August 30th.
Thursday, August 31st.
Friday, September 1st.
Tuesday, September 2nd.
Wednesday, September 3rd
Thursday, September 4th
Friday, September 5th
Saturday, September 6
Sunday, September 7
Monday, September 8th.
Tatenda Tela
hCG for pGEX pcr
Results:
test,
Tuesday, September 9
Inoculation of PCB1c3 (with C4, C5, C7 separately), 13:30.
Eduardo Cepeda.
Wednesday, September 10
Thursday, September 11
Revision of (not grown yet) plates, 12:00.
Eduardo Cepeda.
Inoculaton of D2-GFP, 21:00.
Eduardo Cepeda.
Friday, September 12th
Cleaning of the electroporation cells for the electro-competent cells, 16:00.
Minerva.
Inoculation of cells that grew from the prior day, 18:00.
Cinthya.
Saturday, September 13
Transformation of the D2 and D3 variants for the interlab project, 00:10.
Eduardo Ramirez (2), Mercedes, Claudia, Omar.
Sunday, September 14th.
Miniprep, 17:00.
Mercedes, Eduardo Ramirez (2)
Sterilization of Falcon tubes, LB broth and plastic pearls, 18:30.
Omar
Digestions, 21:00.
Mercedes.
Inoculation of the transformed cells with D2 and D3 variants, 22:00.
Omar.
Monday, September 15
Miniprep of yesterday's inoculations of D2 and D3 variants, 10:00.
Mercedes.
Digestions, 17:30.
Eduardo Ramirez (2).
Inoculation of TOP10 and XL-Blue (Tet), 22:50.
Mercedes.
Tuesday, September 16th.
Transformations of the M13 bacteriophage into the XL-Blue, 10:00.
Mercedes.
Wednesday, September 17
Miniprep 01:15.
Minerva, Eduardo Cepeda.
Digestions (C4, C5, C6, C7 and , 02:53.
Eduardo Cepeda.
PBS buffer, 12:30.
Omar.
Recovery of DNA from Gel, 13:30.
Eduardo Cepeda.
Thursday, September 18
Transformations of C8, C9 and ligation in TOP10 electro-competent, 00:40.
Cinthya, Omar.
Electrophoresis, 01:30.
Mercedes, Eduardo Ramirez (1).
Plates from the transformations, 01:50.
Omar.
Miniprep of PET, 12:30.
Eduardo Cepeda.
Transformation replica of the same ligation as before, 15:30.
Omar.
Preparation of LB broth, 17:00.
All the newbies (Caro, Jesus, Alex, Dany).
Digestion of PET, 19:30.
Claudia.
Friday, September 19
Ligation of PET with all the constructs, 01:19.
Eduardo Cepeda.
Electrophoresis, 03:30.
Eduardo Cepeda.
Miniprep, 8:30.
Mercedes.
Digestions, 13:00.
Mercedes.
Electrophoresis, 15:30.
Mercedes.
Quantification of the fluorescence for the interlab project, 16:30.
Mercedes, Eduardo Ramirez (2).
Group photo taken, 18:00.
Everyone except Mercedes, Eduardo Cepeda and Eduardo Ramirez.
Group video filmed, 18:30.
Everyone.
Transformation of TOP10 cells with PET + (C4, C5, C6, C7), 20:30.
Cinthya, Eduardo Cepeda.
Transformation of TOP10 cells with various repetitions of D2 and D3 (with GFP or RFP), 21:00.
Mercedes, Eduardo Ramirez (2).
Inoculation in Falcon tubes with the ligation of C5 and various others, 22:00.
Omar.
Inoculation in plates of the previous transformed TOP10 cells with PET, 23:00.
Cinthya.
Saturday, September 20
Transformation in electro-competent cells, 01:20.
Mercedes, Eduardo Ramirez.
Minipreps, 18:00.
Mercedes, Omar.
Digestions, 22:00.
Sunday, September 21st.
Electrophoresis, 00:00.
Mercedes.
Inoculations for minipreps, 01:00.
Mercedes.
Minipreps,
Eduardo Cepeda.
Monday, September 22
Miniprep, 08:00.
Mercedes.
Revision of the cell stocks, contamination detected, 09:00.
Mercedes.
Inoculations of C8 and C9, 22:00.
Eduardo Cepeda.
Tuesday, September 23
Preparation of electo-competent cells DH5a, 03:00.
Minerva.
Minipreps for the interlab, 06:00
Mercedes, Minerva.
Transformations for the interlab, 07:30.
Mercedes, Minerva.
Digestions for the interlab, 12:30.
Mercedes.
Miniprep, 13:30.
Eduardo Cepeda.
Electrophoresis for interlab, 19:00.
Eduardo Ramirez (2)
Electrophoresis for the project (C8, C9), 21:00.
Eduardo Ramirez (2)
Wednesday, September 24
Purification of the electrophoresis gel, 01:20.
Mercedes.
Digestions for the interlab, 01:20.
Eduardo Cepeda.
Ligations, 02:00.
Eduardo Cepeda.
Electrophoresis for the interlab, 02:40.
Mercedes.
Inoculations for the interlab, 11:00.
Omar, Mercedes.
Thursday, September 25
Inoculation of XL-Blue, 22:00.
Mercedes.
Infection of the XL-Blue with bacteriophages, 23:00.
Mercedes.
Friday, September 26
Miniprep of the bacteriophage-infected cells, 03:00.
Mercedes.
Preparation of the cells for quantification of fluorescence and OD for the interlab project, from 8:00 to 24:00.
Mercedes.
Purification of the miniprep. 18:00.
Eduardo Ramirez (1), Claudia.
Transformations of C1, C2, Bba_1, Bba_2, and ligations, 19:00.
Cinthya, Omar.
Plating of the transformations, 22:00.
Cinthya.
Saturday, September 27
Inoculation of C1, C2, Bba_1, Bba _2 and ligations, 20:00.
Omar
Sterilization of material, 22:00.
Omar.
Sunday, September 28
Preparation of electro-competent cells, 01:00.
Eduardo Cepeda.
Ligations, 03:00.
Eduardo Cepeda.
Electrophoresis, 12:00.
Eduardo Cepeda.
Minipreps of the inoculations from Saturday, 13:00.
Claudia, Eduardo Ramirez (1).
Recovery of DNA from gel, 15:00.
Claudia.
Monday, September 29
Tuesday, September 30
Wednesday, October 1st.
Minipreps of all constructs, 12:00.
Eduardo.
Digestions of all constructs, 20:30.
Claudia, Jesus.
Miniprep of C2, 21:00.
Jesus.
Thursday, October 2nd.
Collaboration: gave C4 through C9 to the UANL team, 13:30.
Mercedes.
Inoculo XL-Blue, 18:00.
Eduardo Ramirez (2).
Miniprep C8, C10, Bb_1, C6, 16:30.
Eduardo Cepeda, Eduardo Ramirez (2).
Miniprep of phagemides, 18:30.
Eduardo Cepeda.
Friday, October 3rd.
Saturday, October 4th.
Sunday, October 5th.
Monday, October 6th.
Tuesday, October 7th.
Wednesday, October 8th.
Thursday, October 9th.
Friday, October 10th.
Saturday, October 11th.
Sunday, October 12th.
Tatenda Tela
I transformed HCG in PS1C3 into E.coli today
Monday, October 13th.
Tuesday, October 14th.
Sunday, October 15th.
Tatenda Tela
Restriction digests
Ligation
Monday,October 16th.
Eudoxie Bataba
Transformations
Tuesday, October 17th.
Tatenda Tela
Ligation
Wednesday,October 18th.
Tatenda Tela and Cara Jones
Digested miniprepped DNA from glycerol stocks
Gel extraction of the backbones
Thursday, October 19th
Tatenda Tela
Double digests
Ligations
Transformations
Friday, October 20th
Tatenda Tela
Colony pcr
Saturday, October 21st
Tatenda Tela and Aditya Natu
Miniprep colonies
Sunday, October 22nd.
Monday, October 23rd.
Tatenda Tela and Aditya Natu
Colony PCR
Diagnostic restriction digest of colonies
Gel extraction and clean-up
Tuesday, October 24th.
Tatenda Tela
Digested three different plasmids for ligation with HCG. All were PSB1C3 with different inserts.