<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
f
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.
Saturday, September 2nd.
Sunday, September 3rd
Monday, September 4th
Tuesday, September 5th
Wednesday, September 6
Thursday, September 7
Friday, September 8th.
Tatenda Tela
hCG for pGEX pcr
Results:
lane
contents
1
Generuler 1kb plus ladder
2
hCG insert w/ hCG block 5 fwd&rev primers
3
hCG insert w/ hCG block 5 fwd&rev primers
4
hCG insert w/ hCG block 5 fwd&rev primers
5
hCG insert w/ hCG block 5 fwd&rev primers
6
hCG insert w/ hCG block 5 fwd&rev primers
7
hCG insert w/ hCG block 5 fwd&rev primers
8
hCG insert w/ hCG for pGEX fwd&rev primers
9
hCG insert w/ hCG for pGEX fwd&rev primers
10
hCG insert w/ hCG for pGEX fwd&rev primers
11
hCG insert w/ hCG for pGEX fwd&rev primers
12
hCG insert w/ hCG for pGEX fwd&rev primers
13
hCG insert w/ hCG for pGEX fwd&rev primers
14
Generuler 1kb plus ladder
Saturday, September 9
Sunday, September 10
Monday, September 11
Tuesday, September 12
Wednesday, September 13
Cara Jones
Overnight Cultures
Tatenda Tela
Restriction digest and heat inactivation of samples from September 8
Thursday, September 14th.
Tatenda Tela
Miniprep of overnight cultures prepped by Cara the day before
Results:
Sample
Concentration (ng/uL)
A260/A280
1
25
2.0
2
7.5
2.0
3
9.8
1.7
4
8.5
1.8
5
9.5
1.7
Friday, September 15
Saturday, September 16th.
Victoria Radcliffe, Aditya Natu
Midiprep of mamba in pGEX to get pGEx backbone
Sunday, September 17
Monday, September 18
Tuesday, September 19
Tatenda Tela
Digest of Midiprep of mamba in pGEX done by Victoria and Aditya using EcoRI and NotI enzymes
Results:
lane
contents
1
Generuler 1kb plus ladder
2
Mamba in pGEX
3
Mamba in pGEX
4
Mamba in pGEX cut with EcoRI
5
Mamba in pGEX cut with EcoRI
6
7
Mamba in pGEX cut with EcoRI and NotI
8
9
Mamba in pGEX cut with EcoRI and NotI
10
11
Mamba in pGEX cut with EcoRI and PstI
12
Mamba in pGEX cut with EcoRI and PstI
13
14
Wednesday, September 20
Thursday, September 21
Friday, September 22
Saturday, September 23
Sunday, September 24
Monday, September 25
Tuesday, September 26
Wednesday, September 27
Thursday, September 28
Friday, September 29
Saturday, September 30
Sunday, October 1st.
Monday, October 2nd.
Tuesday, October 3rd.
Wednesday, October 4th.
Thursday, October 5th.
Friday, October 6th.
Saturday, October 7th.
Sunday, October 8th.
Monday, October 9th.
Tuesday, October 10th.
Wednesday, October 11th.
Thursday, October 12th.
Tatenda Tela
I transformed HCG in PS1C3 into E.coli today
Friday, October 13th.
Tatenda Tela
I performed PCR and did overnight cultures of 10 colonies
Saturday, October 14th.
Eudoxie Bataba
Objective:PCR diagnostic gel, Miniprep and Restriction DIgest