Minnesota/31 July 2008

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Revision as of 22:32, 31 July 2008

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1. Pick Colonies from BV:Lac/LAMBDA (dual promoter and base vector): Picked colonies and placed BV:dualpromoters in 2mL LB cultures to incubate @ 37C for 8-12 hours. (Also make one 'control' w/ 2mL LB and pipet tip but no colony on it).

2. Plasmid Prep (1) BV:Tet/p22:RFP and (2) BV:Tetpromoter:LAMBDAcIgene: Using QIAmini prep guidelines - prepped 5 samples of BV:TetR/p22:RFP and 5 samples of BV:TetRpromoter:LAMBDAcIgene. Steps: Pour LB culture into tube. Spin tube and remove supernatant. Resuspend pelleted bacterial cells in 250uL Buffer P1 Resuspension buffer. Add 250uL Buffer P2 Lysis buffer and invert 4-6 times. Add 350uL of Buffer N3 Neutralization buffer and invert 4-6 times. Centrifuge for 10 minutes @ 13,000 rpm's. Put supernatants in to spin column, centrifuge for 30-60 seconds (spin column binds to DNA and everything else flows through). Discard flow through. Add 500.0uL of Buffer PB binding buffer, centrifuge for 30-60 seconds, discard flow through. Add 750.0uL Buffer PE wash buffer, centrifuge for 30-60 seconds, discard supernatants and centrifuge for an additional 30-60 seconds. Place spin column in microcentrifuge tube, and elute DNA by adding 50uL Buffer EB elution buffer (10mM Tris-Cl, pH 8.5)let stand for 5 minutes and centrifuge.

NOTE: Last step of Plasmid Prep was performed differently to obtain a higher concentration of DNA. After elution buffer fell through and flow through had DNA in it so repoured that into spin column a second time to spin again for another 60 seconds to extract more DNA.

3. Spectrophotometry: Spec the 10 plasmid prep samples to check for concentration of DNA.

4. Double Digest: Digest Plasmid Prep products. After following procedure below

Parts	10x Buffer	BSA	H20	DNA	RE 1	RE 2	Total
BV + T/P + RFP (1)	5.0uL	0.5uL	2.5uL	40.0uL	1.0uL, Spe1	1.0uL, Pst1	50.0uL
BV + T/P + RFP (2)	5.0uL	0.5uL	2.5uL	40.0uL	1.0uL, Spe1	1.0uL, Pst1	50.0uL
BV + Tetpro. + LAMBDAcI (1)	5.0uL	0.5uL	2.5uL	40.0uL	1.0uL, Spe1	1.0uL, Pst1	50.0uL
BV + Tetpro. + LAMBDAcI (2)	5.0uL	0.5uL	2.5uL	40.0uL	1.0uL, Spe1	1.0uL, Pst1	50.0uL
Terminator	5.0uL	0.5uL	40.5uL	2.0uL	1.0uL, Xba1	1.0uL, Pst1	50.0uL

5. Vector Dephosphorylate: Vector Dephosphorylate the base vector by adding 1uL Antarctic phosphatase and 5uL of Antarctic Phosphatase buffer.

5. Run a gel: Run gel with (1) BV:Tet/p22:RFP and (2) BV:TetRpromoter:LAMBDAcI. Run for 60 minutes @ 80 volts. Results: NO DNA bands found.

6. Plasmid Prep the Lambda/LacI:BV cultures: Follow same steps as step 2.

@@ Line 16: / Line 16: @@
 |'''3. Spectrophotometry:''' Spec the 10 plasmid prep samples to check for concentration of DNA.
 |-
-|'''4. Double Digest:''' Digest Plasmid Prep products.
+|'''4. Double Digest:''' Digest Plasmid Prep products. After following procedure below
+{|border="1" align="left"
+|-
+!| Parts !! 10x Buffer !! BSA !! H20 !! DNA !! RE 1 !! RE 2 !! Total
+|-
+| BV + T/P + RFP (1) || 5.0uL || 0.5uL ||  2.5uL || 40.0uL || 1.0uL, Spe1 || 1.0uL, Pst1 || 50.0uL
+|-
+| BV + T/P + RFP (2) || 5.0uL || 0.5uL ||  2.5uL || 40.0uL || 1.0uL, Spe1 || 1.0uL, Pst1 || 50.0uL
+|-
+| BV + Tetpro. + LAMBDAcI (1) || 5.0uL || 0.5uL ||  2.5uL || 40.0uL || 1.0uL, Spe1 || 1.0uL, Pst1 || 50.0uL
+|-
+| BV + Tetpro. + LAMBDAcI (2) || 5.0uL || 0.5uL ||  2.5uL || 40.0uL || 1.0uL, Spe1 || 1.0uL, Pst1 || 50.0uL
+|-
+| Terminator || 5.0uL || 0.5uL || 40.5uL || 2.0uL || 1.0uL, Xba1 || 1.0uL, Pst1 || 50.0uL
+|}
 |-
 |'''5. Vector Dephosphorylate:''' Vector Dephosphorylate the base vector by adding 1uL Antarctic phosphatase and 5uL of Antarctic Phosphatase buffer.