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<h1>Tuesday, August 15th.</h1> | <h1>Tuesday, August 15th.</h1> | ||
<li> Tori Radcliff</li> | <li> Tori Radcliff</li> | ||
− | <ul> Overlap Extension PCR | + | <ul> Overlap Extension PCR</ul> |
<ul><b>Objective:</b>To combine the four PCR-ed g-blocks of factor-C.</ul> | <ul><b>Objective:</b>To combine the four PCR-ed g-blocks of factor-C.</ul> | ||
<ul><b>Procedure:</b> <a href="https://static.igem.org/mediawiki/2017/1/13/T--Georgia_State--OverlapPCRprotocol.pdf">See attached protocol for details</a></ul> | <ul><b>Procedure:</b> <a href="https://static.igem.org/mediawiki/2017/1/13/T--Georgia_State--OverlapPCRprotocol.pdf">See attached protocol for details</a></ul> |
Revision as of 04:49, 31 October 2017
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Team:Tec-Monterrey/ITESM14 notebook data.html - 2014.igem.org
Tori Radcliff
Tori Radcliff
Tori Radcliff
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.
- Revision of the protocols for next month’s lab-work, 12:00.
- Everyone.
- Revision of human practice ideas and collaborations with other teams, 13:00.
- Everyone.
Wednesday, June 11th.
- Electro-competent cells, 13:00.
- Cinthya
Thursday, June 12th.
- Sponsorships, all day.
- Cinthya.
Friday, June 13th.
- Meeting with advisors, 17:00.
- Calcium-competent cells, 13:00.
- Cinthya.
Saturday, June 14th.
Sunday, June 15th.
Monday, June 16th.
- Electro-competent cells, 14:00.
- Cinthya, Mercedes.
- Minipreps, 16:00.
- Cinthya.
- Electrophoresis, 19:00.
- Cinthya.
- Inoculations, 20:00.
- Mercedes.
Tuesday, June 17th.
Wednesday, June 18th.
- Electro-competent cells, 10:00.
- Cinthya.
- Minipreps, 13:00.
- Mercedes.
- Electrophoresis, 16:00.
- Cinthya.
- Inoculations, 20:00.
- Cinthya.
Thursday, June 19th.
Friday, June 20th.
- Meeting with advisors, 17:00.
Saturday, June 21st.
Sunday, June 22nd.
Monday, June 23rd.
- Calcium-competent cells, 12:00.
- Cinthya.
Tuesday, June 24th.
- Minipreps, 11:00
- Mercedes.
- Electrophoresis, 14:00.
- Mercedes.
- Inoculations. 18:00.
- Mercedes.
Wednesday, June 25nd.
Thursday, June 26th.
- Minipreps, 10:00
- Mercedes.
- Electrophoresis, 13:30.
- Mercedes.
Friday, June 27th.
- Meeting with advisors, 17:00.
Saturday, June 28nd.
Sunday, June 29nd.
Monday, June 30nd.
Tuesday, July 1st.
Wednesday, July 2nd.
Thursday, July 3rd.
Friday, July 4th.
- Minipreps, 12:00.
- Mercedes.
- Practice: Ligations, 15:00.
- Eduardo Ramirez (1).
- Practice: Electrophoresis, 17:00.
- Eduardo Ramirez (1)
Wednesday, July 5th.
Thursday, July 6th.
Friday, July 7th.
- 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
- Equipment:UV-Crosslinker
- Procedure:See attached procedure.
- 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.
![](https://static.igem.org/mediawiki/2017/c/c5/T--Georgia_State--APTest.png)
Tuesday, July 8th.
Wednesday, July 9th.
Thursday, July 10th.
- Practice: Transformation of calcium-competent cells, 11:00,
- Eduardo Ramirez (1), Claudia.
- Inoculation on LB-agar plates, 13:00.
- Claudia.
- Laboratory summer cleaning, 15:00.
- Claudia.
Friday, July 11th.
Saturday, July 12th.
Sunday, July 13th.
Monday, July 14th.
Tuesday, July 15th.
- Practice: taking DNA out of the iGEM plates, 13:00.
- Claudia.
- Transformation with calcium-competent cells, 13:30.
- Claudia.
- Inoculations, 15:00.
- Claudia.
Wednesday, July 16th.
- Minipreps, 12:00.
- Eduardo Ramirez (1).
- Ligations, 15:00.
- Eduardo Ramirez (1).
- Electrophoresis, 18:00.
- Practice: DNA recovery from gel, 20:00.
- Eduardo Ramirez (1)
Thursday, July 17th.
Friday, July 18th.
Saturday, July 19th.
- Meeting with advisors to talk about gene problems and doubts, 17:00.
Sunday, July 20th.
Monday, July 21st.
Tuesday, July 22nd.
- Minipreps, 11:00.
- Claudia.
- Ligations, 15:00.
- Claudia.
- Electrophoresis, 19:00.
- Claudia
- Practice: DNA recovery from gel, 21:00.
- Claudia.
Wednesday, July 23rd.
Thursday, July 24th.
Friday, July 25th.
Saturday, July 26th.
- Meeting with advisors, 17:00.
Monday, July 29th.
- Meeting to plan the semester’s activities, 14:00.
- Schedule of people in the lab.
- Ways to integrate other students.
- Events to talk about iGEM.
Sunday, July 27th.
Monday, July 28th.
Tuesday, July 29th.
Wednesday, July 30th.
Thursday, July 31st.
Tuesday,August 1st.
- 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
- Equipment:Thermocycler
- Procedure:See attached procedure.
- Results:
![]() | ![]() |
Simulated gel using SnapGene | 1% Agarose gel, experimental results |
Wednesday, August 2nd.
Thursday, August 3rd.
- Objective:To double digest both the PSB1C3 backbone and our hCG PCR product
- Materials:DNA of ECFP-PCB1C3 (102 ng/ul) and hCG PCR product, Restriction enzymes: NotI, EcoRI, XbaI, PstI,SpeI, Green Fastdigest Buffer (10x), and Nuclease free H20
- Equipment:Thermocycler
- Procedure:See attached procedure.
- Results:
- 1. 1Kb New England Biolabs Ladder
- 2. hCG uncut
- 3. hCG cut with EcoR1 and Pst1
- 4. hCG cut with Xba1 and Spe1
- 5. ECFP cut with EcoR1 and Pst1
- 6. ECFP cut with Xba1 and Spe1
![]() | ![]() |
Simulated gel using SnapGene | The Wells Contain the following:
|
Friday August 4th.
- Objective: Clean used GST resin so that it may be used in future protein purifications.
- Materials: GST Resin, 70% Ethanol, GST Elution Buffer
- Equipment: Centrifuge,Vortexer, Micropipette,Eppendorf tubes/Falcon tubes, Spectrophotometer, and Cuvettes
- Procedure:See attached procedure for buffer recipes and instructions.
- Results:
Sample | Absorbance @ 280 nm |
---|---|
Clean GST Elution Buffer (Blank) | 0 |
Elution 1 | 0.667 |
Elution 2 | 0.036 |
Saturday, August 5th.
- 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
- Equipment:Thermocycler
- Procedure:See attached procedure.
- Results:
![]() | Since the diagnostic gel was a success the PSB1C backbone was extracted from the Lt 10 plasmid using an E-gel. |
Sunday, August 6th.
Monday, August 7th.
Tuesday, August 8th.
Wednesday, August 9th.
- Ligation of hCG to PSB1C3
Thursday, August 10th.
- Transformation of hCG-PSB1C3 ligation and overnight culture
Friday, August 11th.
- Colony PCR of hCG-PSB1C3 colonies
Saturday, August 12th.
Sunday, August 13th.
- Overnight culture of hCG-PSB1C3 colonies
Monday, August 14th.
- 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.
- Procedure: See attached protocol.
- Results:
![]() |
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.
- Overlap Extension PCR
- Objective:To combine the four PCR-ed g-blocks of factor-C.
- Procedure: See attached protocol for details
- Results:
- Tori Radcliff
- Tori Radcliff
- Tori Radcliff
- Apoptosis kit arrived!, 14:00.
- Accutase arrived!, 14:00.
- Genes arrived!, 14:00.
- Preparation of LB and LB-agar media, 13:30.
- Eduardo Ramirez (1).
- Preparation of the plaques for the transformation, 17:00.
- Eduardo Cepeda.
- HyperLadder 50bp arrived!, 17:10
- HLA arrived!, 17:10:
- Transformation of C1 through C7 in TOP10, 19:00.
- Minerva.
- Transformation of GFP and RFP in TOP10, 21:30.
- Mercedes.
- Inoculation of plates with transformed colonies from yesterday, 12:00.
- Minerva.
- Transformations of GFP Amp, RFP Cam, D1 Kn, D2 Cam, D3 Amp, 19:30.
- Eduardo Ramirez (2).
- Miniprep of the grown transformed cells from yesterday (C4, C5, C6, C7, D2, GFP, D1, BBa_1), 21:00.
- Eduardo Cepeda.
- Digestions of the last minipreps, 02:00.
- Eduardo Cepeda.
- Electrophoresis, 03:30.
- Eduardo Cepeda.
- Preparation of electro-competent TOP10 cells, 08:00.
- Newbies: Alex, Danny, Chuy, Caro.
- Preparation of stock cells (C4, C5, C6, C7, D2, GFP, D1, BBa_1), 18:30.
- Mercedes.
- Ligations of the C4, C5, C6, C7 with the pcb1c3, 20:30.
- Eduardo Cepeda.
- Inoculation of TOP10 for electro-competent cells tomorrow and D2 cells, 01:25.
- Mercedes.
- Preparation of the tickets for the cake raffle (fundraising), 12:00.
- Cinthya, Omar, Eduardo Ramirez (1), Eduardo Cepeda, Mercedes.
- Miniprep, 14:00.
- Mercedes and Eduardo Cepeda with special guest: Ana Cristina.
- Talk with Professor Cardineau about the legislation proposal, 17:00.
- Omar.
- Preparations of electro-competente TOP10 cells, 17:30.
- Eduardo Ramirez (2), Mercedes.
- Digestions, 19:00.
- Eduardo Cepeda.
- Inoculation of LB-agar plaques, 21:00.
- Cynthia.
- Inoculation of PCB1c3 (with C4, C5, C7 separately), 13:30.
- Eduardo Cepeda.
- Revision of (not grown yet) plates, 12:00.
- Eduardo Cepeda.
- Inoculaton of D2-GFP, 21:00.
- Eduardo Cepeda.
- 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.
- Transformation of the D2 and D3 variants for the interlab project, 00:10.
- Eduardo Ramirez (2), Mercedes, Claudia, Omar.
- 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.
- 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.
- Transformations of the M13 bacteriophage into the XL-Blue, 10:00.
- Mercedes.
- 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.
- 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.
- 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.
- Transformation in electro-competent cells, 01:20.
- Mercedes, Eduardo Ramirez.
- Minipreps, 18:00.
- Mercedes, Omar.
- Digestions, 22:00.
- Electrophoresis, 00:00.
- Mercedes.
- Inoculations for minipreps, 01:00.
- Mercedes.
- Minipreps,
- Eduardo Cepeda.
- Miniprep, 08:00.
- Mercedes.
- Revision of the cell stocks, contamination detected, 09:00.
- Mercedes.
- Inoculations of C8 and C9, 22:00.
- Eduardo Cepeda.
- 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)
- 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.
- Inoculation of XL-Blue, 22:00.
- Mercedes.
- Infection of the XL-Blue with bacteriophages, 23:00.
- Mercedes.
- 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.
- Inoculation of C1, C2, Bba_1, Bba _2 and ligations, 20:00.
- Omar
- Sterilization of material, 22:00.
- Omar.
- 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.
- Minipreps of all constructs, 12:00.
- Eduardo.
- Digestions of all constructs, 20:30.
- Claudia, Jesus.
- Miniprep of C2, 21:00.
- Jesus.
- 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.
- Digestions of C1, C2 and PET variants of the other constructs, 12:00.
- Eduardo Cepeda.
- Minipreps, 12:00.
- Claudia.
- Electrophoresis, 16:00.
- Eduardo Cepeda.
- Inoculations, 20:00.
- Mercedes.
- Western Blot, 01:00.
- Minerva.
- Minipreps, 00:30.
- Eduardo Cepeda.
- Electrophoresis, 04:00.
- Eduardo Cepeda.
- Inoculations, 18:00.
- Eduardo Cepeda.
- Minipreps for cells with flippase, 12:00.
- Eduardo Cepeda.
- Stock of cells with flippase, 12:00.
- Mercedes.
- Digestions for flippase, 16:00.
- Eduardo Cepeda.
- Electrophoresis, 18:00.
- Eduardo Cepeda.
- Digestions of Bba_1, Bba_2 and Bba_3, 08:00.
- Eduardo Cepeda.
- Digestion of the modified phage, 09:00.
- Mercedes.
- Electrophoresis, 12:00.
- Eduardo Ramirez (2).
- Ligation of the phages back to circle form, 13:00.
- Mercedes.
- Inoculations for constructs stocks, 20:00.
- Mercedes.
- Pictures taken for the wiki, 13:00.
- Everyone.
- PCR, 16:00.
- Eduardo Cepeda.
- Stocks, 18:00.
- Mercedes.
- Electrophoresis, 20:00.
- Eduardo Cepeda.
- PCR for Red Lambda confirmation, 13:00.
- Eduardo Cepeda.
- Electrophoresis, 15:00.
- Mercedes.
- Digestions, 16:00.
- Mercedes.
- TAE buffer preparation, 20:00.
- Newbies: Alejandro, Danny.
- Western Blott, 21:00.
- Minerva, Claudia.
- Work on wiki
- Sebastián Valdivieso, Eduardo Zardain, Eduardo Ramírez
- Work on wiki
- Sebastián Valdivieso, Eduardo Zardain, Eduardo Ramírez
- Work on wiki
- Sebastián Valdivieso, Eduardo Zardain, Eduardo Ramírez
- Work on wiki, its wiki-freeze day!!!
- Sebastián Valdivieso, Eduardo Zardain, Eduardo Ramírez
![]() | ![]() | ||
Abbreviation |
Construct |
Gene |
Resistance |
LPP |
C1 |
LPP Red Lambda |
Amp |
MSBB |
C2 |
Red Lambda |
Kn + Amp |
H-GFP |
C4 |
GFP |
Amp |
C-Apop |
C5 |
Apoptine |
Amp |
H-SiRNA 1 |
C6 |
SiRNA 1 |
Amp |
H-SiRNA 2 |
C7 |
SiRNA 2 |
Amp |
H-SiRNA 3 |
C8 |
SiRNA 3 |
Amp |
H-SiRNA-Apop |
C9 |
SiRNA + Apoptine |
Amp |
BBa_1 |
Biobrick |
Cam |
|
BBa_2 |
Biobrick |
Cam |
|
BBa_3 |
Biobrick |
Peptide + p3 |
Cam |