Minnesota/24 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.

@@ Line 59: / Line 59: @@
 {|border="1" align="left"
 |-
-!| Parts !! 10x Buffer !! H20 !! Base Vector !! Insert DNA !! T4 DNA Ligase !! Total Volume !!
+!| 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
@@ Line 75: / Line 75: @@
 | 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.