Minnesota/29 July 2008

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Revision as of 22:40, 29 July 2008

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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:

 PRIMER MIXES

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

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 indicted the presence of both PCR products (Lac/LAMBDA and TetR/p22) and digest products (LAMBDAcI and RFP).

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.

@@ Line 3: / Line 3: @@
 |'''[[Team:Minnesota/NotebookComparator|  Back to Notebook Home]]'''
 |-
-|'''[[Minnesota/28 July 2008|Go to Previous Day (July 28)]]'''|| width=158|'''[[Minnesota/29 July 2008|Go to Next Day (July 29)]]'''
+|'''[[Minnesota/28 July 2008|Go to Previous Day (July 28)]]'''|| width=158|'''[[Minnesota/30 July 2008|Go to Next Day (July 30)]]'''
 |}
 {|
 |-
-|'''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.
+|''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''
+{|border="1" align="left"
 |-
-|'''2. Double Digest:''' Digested two things only - LAMBDAcI and RFP. Follow the table 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''
+{|border="1" align="left"
+|-
+!| 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:
+{|border="1" align="left"
+|-
+!| 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 indicted the presence of both PCR products (Lac/LAMBDA and TetR/p22) and digest products (LAMBDAcI and RFP).
+|-
+|'''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:
+{|border="1" align="left"
+|-
+!| 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.