| 1. Spec the plasmid prep products: Place 10 samples into 10 separate plate wells. 11 wells total were used: 1st well was the control so it had 36uL of distilled water and 4uL of elution buffer (since want everything except DNA to be control group), then 2-11 wells had 1-10 plasmid prep products. 2-11 wells had 36uL of distilled water added and 4uL of plasmid prep products. Results:
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| a. (1) Lac I #1 = [DNA] ng/uL is 45, [DNA] ug/uL is 0.045 HIGH/GOOD
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| b. (2) Lac I = [DNA] ng/uL is 35, [DNA] ug/uL is 0.035 HIGH/GOOD
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| c. (3) Lac I = [DNA] ng/uL is 35, [DNA] ug/uL is 0.035 HIGH/GOOD
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| d. (4) BV:Lac #2 = [DNA] ng/uL is 10, [DNA] ug/uL is 0.001 LOW/POOR
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| e. (5) BV:Tet #1 = [DNA] ng/uL is 0, [DNA] ug/uL is 0.00 NONE/BAD
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| f. (6) BV:Tet #2 = [DNA] ng/uL is 5, [DNA] ug/uL is 0.005 LOW/POOR
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| g. (7) BV:Tet/p22 #1 = [DNA] ng/uL is 5, [DNA] ug/uL is 0.005 LOW/POOR
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| h. (8) BV:Tet/p22 #1 = [DNA] ng/uL is 5, [DNA] ug/uL is 0.005 LOW/POOR
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| i. (9) BV:Tet/p22 #2 = [DNA] ng/uL is 335, [DNA] ug/uL is 0.335 HIGH/GOOD
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| j. (10) BV:Tet/p22 #2 = [DNA] ng/uL is 15, [DNA] ug/uL is 0.015 LOW/POOR
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| 2. Digest Rxn: Using #'s: 1, 2, 6, 9, and 10 from above. Also using: p22cII, LAMBDAcI, and RFP from different plasmid prep days. After following table below, allow to incubate for 2 hours. Heat inactivate enzymes @ 65C for 15 minutes in water bath. Follow the table below:
| Parts | 10x Buffer | BSA | DNA | RE 1 | RE 2 | H20
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| BV:Lac I (1) | 5.0uL | 0.5uL | 22.0uL | Spe1, 1.0uL | Pst1, 1.0uL | 20.5uL
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| BV:Lac I #2 (2) | 5.0uL | 0.5uL | 28.5uL | Spe1, 1.0uL | Pst1, 1.0uL | 14.0uL
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| BV:Tet (6) | 5.0uL | 0.5uL | 35.0uL | Spe1, 1.0uL | Pst1, 1.0uL | 7.5uL
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| BV:Tet/p22 #1 (9) | 5.0uL | 0.5uL | 3.0uL | Spe1, 1.0uL | Pst1, 1.0uL | 39.5uL
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| BV:Tet/p22 #2 (10) | 5.0uL | 0.5uL | 33.0uL | Spe1, 1.0uL | Pst1, 1.0uL | 9.5uL
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| p22cII | 5.0uL | 0.5uL | 5.0uL | Xba1, 1.0uL | Pst1, 1.0uL | 37.5uL
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| LAMBDAcI | 5.0uL | 0.5uL | 7.0uL | Xba1, 1.0uL | Pst1, 1.0uL | 35.5uL
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| RFP | 5.0uL | 0.5uL | 4.0uL | Xba1, 1.0uL | Pst1, 1.0uL | 38.5uL
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| 3. Vector Dephosphorylate: Dephosphorylate the base vectors of the digested products. After following the table below, allow to incubate for 30 minutes @37C. Place samples in water bath @ 65C to deactivate the enzyme. Place samples in freezer @ -20C O/N to continue Ligation. Follow the table below:
| Parts | Antarctic Phosphatase | Antarctic Phosphatase Buffer
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| BV:LacI #1 (1) | 1.0uL | 5.0uL
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| BV:LacI #2 (2) | 1.0uL | 5.0uL
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| BV:Tet (6) | 1.0uL | 5.0uL
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| BV:Tet/p22 #1 (9) | 1.0uL | 5.0uL
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| BV:Tet/p22 #2 (10) | 1.0uL | 5.0uL
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|
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