Minnesota/29 July 2008

From 2008.igem.org

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|'''[[Team:Minnesota/NotebookComparator|  Back to Notebook Home]]'''
|'''[[Team:Minnesota/NotebookComparator|  Back to Notebook Home]]'''
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|'''[[Minnesota/28 July 2008|Go to Previous Day (July 28)]]'''|| width=158|'''[[Minnesota/29 July 2008|Go to Next Day (July 29)]]'''
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|'''[[Minnesota/28 July 2008|Go to Previous Day (July 28)]]'''|| width=158|'''[[Minnesota/30 July 2008|Go to Next Day (July 30)]]'''
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|'''1. Used PCR to produce more lambdacI gene with the RBS ligated to the 5' end''' Once the PCR procedure had been successfully completed, the PCR product and RFP were redigested following exactly the procedure used yesterday.  
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|'''1. PCR Reaction: PCR LacI/LAMBDAcI and TetR/p22mnt dual promoters:''' PCR'ed these two dual promoters to remove RBS so that it can be submitted to iGEM as a single part. Follow the table below:
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|- 
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|''PRIMER MIXES below...''
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{|border="1" align="left"
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|-
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!| Dual Promoters !! prim 1F !! prim 2R !! H20
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|-
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| LacI/LAMBDAcI || 1.5uL || 1.5uL || 12.0uL
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|-
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| TetR/p22mnt || 1.5uL || 1.5uL || 12.0uL
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|}
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|-
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| ''PCR: Dual Promoters below...''
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{|border="1" align="left"
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|-
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!| Parts !! 5x HF Buffer !! dNTP's !! primer mix !! DNA template !! phusion hot start polymerase !! H20 !! Total
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|-
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| Lac/LAMBDA || 40.0uL || 4.0uL || 10.0uL || 40.0uL || 2.0uL || 104.0uL || 200.0uL
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|-
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| TetR/p22mnt || 40.0uL || 4.0uL || 10.0uL || 29.0uL || 2.0uL || 115.0uL || 200.0uL
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|-
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| L/L control || 10.0uL || 1.0uL || 2.5uL || 0 || 0.5uL || 36.0uL
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|-
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| T/P control || 10.0uL || 1.0uL || 2.5uL || 0 || 0.5uL || 36.0uL
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|}
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|-
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|'''2. Different PCR reaction: Used PCR to produce more lambdacI gene with the RBS ligated to the 5' end''' Once the PCR procedure had been successfully completed, the PCR product (LAMBDAcI) was then used in the double digest along with RFP.
 +
|-
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|'''3. Double Digest:''' Digested two things only - LAMBDAcI and RFP. Used higher concentrations of parts' DNA to attempt a better spec'ing result. Incubate digest products for 2 hours. Heat inactivate enzymes for 15 minutes in a 65C water bath. Follow the table below:
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{|border="1" align="left"
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|-
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!| Parts !! 10x Buffer !! BSA !! DNA !! RE 1 !! RE 2 !! H20
 +
|-
 +
| LAMBDAcI || 5.0uL || 0.5uL || 40.0uL || Xba1, 1.0uL || Pst1, 1.0uL || 2.5uL
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|-
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| RFP || 5.0uL || 0.5uL || 18.0uL || Xba1, 1.0uL || Pst1, 1.0uL || 24.5uL
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|}
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|-
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|'''3. Gel electrophoresis:''' Ran a gel to check if any DNA or correct DNA is in digested products. The gel confirmed presence of digest products (LAMBDAcI and RFP) of the correct size Lanes (L -> R) are 1kb ladder, two lanes of lambda cI digest, two lanes of RFP digest, and a second 1 kb ladder. The three bands in the RFP sample are believed to be undigested base vector + RFP, plasmid backbone, and the RFP gene. Digested Lambda cI gene and RFP gene (lowest bands in picture) were cut out for purification.
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[[Image:Gel7.29.08.jpg|500px||center|thumb|Electrophoretic gel run on 7.29.08]]
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|-
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|'''4. Gel purified:''' Purify the DNA bands out of the gel by following previously performed purification techniques to 'purify' or extract the DNA from the gel.
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|-
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|'''5. Ligation:''' Ligate (1) RFP + T/P + BV and (2) LAMBDAcI + TetR promoter + BV. In #1, the BV and TetR/p22mnt dual promoters are already there - are just ligating into that the RFP. In #2, the BV and TetR promoter are already there - are just ligating the LAMBDAcI onto that. After ligated, allow to incubate 30 minutes then heat shock enzyme for 15 minutes @ 65C in a water bath. Then transform into competent cells. Follow the table below:
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{|border="1" align="left"
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|-
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!| Parts !! 10x Buffer !! H20 || Base Vector 1|| Base Vector 2 || T4 DNA ligase || Total
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|-
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| RFP + T/P + BV || 6.0uL || 0 || 4.0uL || 44.0uL || 6.0uL || 60.0uL
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|-
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| LAMBDAcI + Tet promoter + BV || 6.0uL || 0 || 4.5uL || 23.5uL || 6.0uL || 60.0uL
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|}
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|-
|-
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|'''2. Double Digest:''' Digested two things only - LAMBDAcI and RFP. Follow the table below:
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|'''6. Transform ligated products into competent cells:''' Allow to incubate O/N @37C with shaking at 220rpm's.

Latest revision as of 21:04, 6 August 2008

Back to Notebook Home
Go to Previous Day (July 28)Go to Next Day (July 30)
1. PCR Reaction: PCR LacI/LAMBDAcI and TetR/p22mnt dual promoters: PCR'ed these two dual promoters to remove RBS so that it can be submitted to iGEM as a single part. Follow the table below:
PRIMER MIXES below...


Dual Promoters prim 1F prim 2R H20
LacI/LAMBDAcI 1.5uL 1.5uL 12.0uL
TetR/p22mnt 1.5uL 1.5uL 12.0uL
PCR: Dual Promoters below...


Parts 5x HF Buffer dNTP's primer mix DNA template phusion hot start polymerase H20 Total
Lac/LAMBDA 40.0uL 4.0uL 10.0uL 40.0uL 2.0uL 104.0uL 200.0uL
TetR/p22mnt 40.0uL 4.0uL 10.0uL 29.0uL 2.0uL 115.0uL 200.0uL
L/L control 10.0uL 1.0uL 2.5uL 0 0.5uL 36.0uL
T/P control 10.0uL 1.0uL 2.5uL 0 0.5uL 36.0uL


2. Different PCR reaction: Used PCR to produce more lambdacI gene with the RBS ligated to the 5' end Once the PCR procedure had been successfully completed, the PCR product (LAMBDAcI) was then used in the double digest along with RFP.
3. Double Digest: Digested two things only - LAMBDAcI and RFP. Used higher concentrations of parts' DNA to attempt a better spec'ing result. Incubate digest products for 2 hours. Heat inactivate enzymes for 15 minutes in a 65C water bath. Follow the table below:


Parts 10x Buffer BSA DNA RE 1 RE 2 H20
LAMBDAcI 5.0uL 0.5uL 40.0uL Xba1, 1.0uL Pst1, 1.0uL 2.5uL
RFP 5.0uL 0.5uL 18.0uL Xba1, 1.0uL Pst1, 1.0uL 24.5uL
3. Gel electrophoresis: Ran a gel to check if any DNA or correct DNA is in digested products. The gel confirmed presence of digest products (LAMBDAcI and RFP) of the correct size Lanes (L -> R) are 1kb ladder, two lanes of lambda cI digest, two lanes of RFP digest, and a second 1 kb ladder. The three bands in the RFP sample are believed to be undigested base vector + RFP, plasmid backbone, and the RFP gene. Digested Lambda cI gene and RFP gene (lowest bands in picture) were cut out for purification.
Electrophoretic gel run on 7.29.08
4. Gel purified: Purify the DNA bands out of the gel by following previously performed purification techniques to 'purify' or extract the DNA from the gel.
5. Ligation: Ligate (1) RFP + T/P + BV and (2) LAMBDAcI + TetR promoter + BV. In #1, the BV and TetR/p22mnt dual promoters are already there - are just ligating into that the RFP. In #2, the BV and TetR promoter are already there - are just ligating the LAMBDAcI onto that. After ligated, allow to incubate 30 minutes then heat shock enzyme for 15 minutes @ 65C in a water bath. Then transform into competent cells. Follow the table below:


Parts 10x Buffer H20 Base Vector 1 Base Vector 2 T4 DNA ligase Total
RFP + T/P + BV 6.0uL 0 4.0uL 44.0uL 6.0uL 60.0uL
LAMBDAcI + Tet promoter + BV 6.0uL 0 4.5uL 23.5uL 6.0uL 60.0uL
6. Transform ligated products into competent cells: Allow to incubate O/N @37C with shaking at 220rpm's.
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