Minnesota/24 July 2008

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|'''5. Gel electrophoresis:''' After heat inactivating the enzyme @ 65C for 15 minutes, we ran a gel with the BV and double digest proucts.  
|'''5. Gel electrophoresis:''' After heat inactivating the enzyme @ 65C for 15 minutes, we ran a gel with the BV and double digest proucts.  
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|-
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|'''6. Gel Purified:''' Cut out bands from gel and purified those bands (which contain the DNA wanted).  
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|'''6. Gel Purified:''' Cut out bands from gel and purified those bands (which contain the DNA wanted). Follow QIA protocol: (1) Excise the DNA fragment from the agarose gel with a clean blade, (2) Place gel fragments in designated 2.0mL tubes, (3) Add 1.0mL  of Buffer QG to gel in 2.0mL tubes - allow to solubilize (dissolve) for 10 minutes @ 50C and mix by vortexing, (4) After the gel slice has dissolved completely add 1.0mL of 100% isopropanol to the samples and mix, (5) Place samples into a QIAquick spin column to bind DNA and centrifuge for 1 minute, (6) Discard flow through and place spin column back in same collection tube, (7) Add 0.5mL of Buffer QG to spin column and centrifuge for 1 minute, (8) Discard flow through and add 0.75mL of Buffer PE to spin column and centrifuge for 1 minute, (9) discard the flow through and centrifuge for additional 1 minute @ 10,000rcf, (10) Place column into a clean 1.5mL microcentrifuge tube, (11) To elute DNA add 50uL of Buffer EB (10 mM Tris-Cl, pH 8.5) to the center of QIAquick membrane and centrifuge for 1 minute. The flow through is the purified DNA, which will be used in spectrophotometry.
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|-
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|'''7. Spetrophotometry:''' Spec the purified gel DNA to measure concentration of DNA in eight parts.
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|'''7. Spetrophotometry:''' Spec the purified gel DNA to measure concentration of DNA in eight parts. 9 wells have 36.0uL of distilled water, the 1st well will have 4.0uL of buffer EB added as the control, the following 8 wells will have 4.0uL of the designated DNA from the purified gel.
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|-
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|'''8. Ligations:''' Ligate the spec'ed purified DNA samples. 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 !! Insert DNA !! T4 DNA Ligase !! Total Volume
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|-
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| BV + TetR 1 || 4.0uL || 13.0uL || 5.0uL || 17.0uL || 1.0uL || 40.0uL
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|-
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| BV + TetR 2 || 4.0uL || 6.0uL || 5.0uL || 24.0uL || 1.0uL || 40.0uL
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|-
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| BV + LacI 1 || 4.0uL || 10.0uL || 5.0uL || 20.0uL || 1.0uL || 40.0uL
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|-
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| BV + LacI 2 || 4.0uL || 15.0uL || 5.0uL || 15.0uL || 1.0uL || 40.0uL
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|-
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| BV + Lac/LAMBDA || 4.0uL || 17.0uL || 5.0uL || 13.0uL || 1.0uL || 40.0uL
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|-
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| BV + TetR/p22 1 || 4.0uL || 13.0uL || 5.0uL || 17.0uL || 1.0uL || 40.0uL
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|-
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| BV + TetR/p22 2 || 4.0uL || 6.0uL || 5.0uL || 24.0uL || 1.0uL || 40.0uL 
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|}
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|-
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|'''9. Transform:''' Heat inactivate the T4 DNA ligase in the ligated products by heating @ 65C for 15 minutes in a water bath. Transform the ligated products. Will be transforming 7 things instead of 8, because the base vector is in theory ligated or attached to a particular part in the ligation step. Will transform into TOP 10 Cells the following: (1) BV:TetR/p22 1, (2) BV:TetR/p22 2, (3) BV:Lac/LAMBDA, (4) BV:LacI 1, (5) BV:LacI 2, (6) BV:TetR 1, (7) BV:TetR 2. The following steps will take place for transforming 1-7:
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|-
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|a. Thaw TOP10 cells and incubate transformant (1-7) DNA on ice
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|-
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|b. Combine 50uL thawed cells with 50uL DNA and mix gently.
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|-
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|c. Incubate samples on ice for 20 minutes.
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|-
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|d. Heat shock cells in a 42C water bath for 1 minute.
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|-
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|e. Incubate samples on ice for 5 minutes.
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|-
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|f. Recover cells in 1.0mL 2xTy media, place cultures in incubator @ 37C for 1 hour with shaking @ 220rpm.
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|-
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|'''10. Plate transformed TOP10 Cultures:''' Plate approximately 150uL of culture on kanamycin resistant plates O/N @ 37C.

Latest revision as of 00:17, 25 July 2008

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1. Base Vector Plasmid Maxi Prep Base Vector: Grew another culture of different base vector colonies, so now need to plasmid prep the base vector so the DNA/plasmid will be extracted from the rest of the cell.
2. Spectrophotometry: Excellent results from spec'ing new base vector plasmid. Very high concentration of DNA, which was 430 ng/uL.
3. Base Vector & Promoter Double Digest: 50uL Reaction Mixture. Digest incubation for 2 hours @ 37C. Heat inactivate enzyme for 15 minutes @ 65C in a water bath. Follow the table below for pre-incubation steps:


Parts 10x Buffer BSA H20 DNA from parts RE 1 RE 2 [DNA] ng/uL
BV 5.0uL 0.5uL 30.8uL 11.7uL EcoRI, 1.0uL Pst1, 1.0uL 100.62 ng/uL
TetR Pro. 1 5.0uL 0.5uL 35.5uL 7.0uL EcoRI, 1.0uL Pst1, 1.0uL 23.8 ng/uL
Tet R Pro. 2 5.0uL 0.5uL 35.5uL 7.0uL EcoRI, 1.0uL Pst1, 1.0uL 23.8 ng/uL
LacI Pro. 1 5.0uL 0.5uL 41.5uL 1.0uL EcoRI, 1.0uL Pst1, 1.0uL 168.0 ng/uL
LacI Pro. 2 5.0uL 0.5uL 41.5uL 1.0uL EcoRI, 1.0uL Pst1, 1.0uL 168.0 ng/uL
Lac/Lam 1 5.0uL 0.5uL 28.5uL 14.0uL EcoRI, 1.0uL Pst1, 1.0uL 9.8 ng/uL
Tet/p22 1 5.0uL 0.5uL 34.0uL 8.5uL EcoRI, 1.0uL Pst1, 1.0uL 20.4 ng/uL
Tet/p22 2 5.0uL 0.5uL 34.0uL 8.5uL EcoRI, 1.0uL Pst1, 1.0uL 20.4 ng/uL
4. Vector Dephosphorylation: After the digested products' enzyme has been heat inactivated, we dephosphorylated the base vector so the cut ends wouldn't religate. Follow the table below:


Parts Antarctic Phosphatase Antarctic Phosphatase Buffer
BV 1.0uL 5.1uL
5. Gel electrophoresis: After heat inactivating the enzyme @ 65C for 15 minutes, we ran a gel with the BV and double digest proucts.
6. Gel Purified: Cut out bands from gel and purified those bands (which contain the DNA wanted). Follow QIA protocol: (1) Excise the DNA fragment from the agarose gel with a clean blade, (2) Place gel fragments in designated 2.0mL tubes, (3) Add 1.0mL of Buffer QG to gel in 2.0mL tubes - allow to solubilize (dissolve) for 10 minutes @ 50C and mix by vortexing, (4) After the gel slice has dissolved completely add 1.0mL of 100% isopropanol to the samples and mix, (5) Place samples into a QIAquick spin column to bind DNA and centrifuge for 1 minute, (6) Discard flow through and place spin column back in same collection tube, (7) Add 0.5mL of Buffer QG to spin column and centrifuge for 1 minute, (8) Discard flow through and add 0.75mL of Buffer PE to spin column and centrifuge for 1 minute, (9) discard the flow through and centrifuge for additional 1 minute @ 10,000rcf, (10) Place column into a clean 1.5mL microcentrifuge tube, (11) To elute DNA add 50uL of Buffer EB (10 mM Tris-Cl, pH 8.5) to the center of QIAquick membrane and centrifuge for 1 minute. The flow through is the purified DNA, which will be used in spectrophotometry.
7. Spetrophotometry: Spec the purified gel DNA to measure concentration of DNA in eight parts. 9 wells have 36.0uL of distilled water, the 1st well will have 4.0uL of buffer EB added as the control, the following 8 wells will have 4.0uL of the designated DNA from the purified gel.
8. Ligations: Ligate the spec'ed purified DNA samples. Follow the table below:


Parts 10x Buffer H20 Base Vector Insert DNA T4 DNA Ligase Total Volume
BV + TetR 1 4.0uL 13.0uL 5.0uL 17.0uL 1.0uL 40.0uL
BV + TetR 2 4.0uL 6.0uL 5.0uL 24.0uL 1.0uL 40.0uL
BV + LacI 1 4.0uL 10.0uL 5.0uL 20.0uL 1.0uL 40.0uL
BV + LacI 2 4.0uL 15.0uL 5.0uL 15.0uL 1.0uL 40.0uL
BV + Lac/LAMBDA 4.0uL 17.0uL 5.0uL 13.0uL 1.0uL 40.0uL
BV + TetR/p22 1 4.0uL 13.0uL 5.0uL 17.0uL 1.0uL 40.0uL
BV + TetR/p22 2 4.0uL 6.0uL 5.0uL 24.0uL 1.0uL 40.0uL
9. Transform: Heat inactivate the T4 DNA ligase in the ligated products by heating @ 65C for 15 minutes in a water bath. Transform the ligated products. Will be transforming 7 things instead of 8, because the base vector is in theory ligated or attached to a particular part in the ligation step. Will transform into TOP 10 Cells the following: (1) BV:TetR/p22 1, (2) BV:TetR/p22 2, (3) BV:Lac/LAMBDA, (4) BV:LacI 1, (5) BV:LacI 2, (6) BV:TetR 1, (7) BV:TetR 2. The following steps will take place for transforming 1-7:
a. Thaw TOP10 cells and incubate transformant (1-7) DNA on ice
b. Combine 50uL thawed cells with 50uL DNA and mix gently.
c. Incubate samples on ice for 20 minutes.
d. Heat shock cells in a 42C water bath for 1 minute.
e. Incubate samples on ice for 5 minutes.
f. Recover cells in 1.0mL 2xTy media, place cultures in incubator @ 37C for 1 hour with shaking @ 220rpm.
10. Plate transformed TOP10 Cultures: Plate approximately 150uL of culture on kanamycin resistant plates O/N @ 37C.
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