Template:Team:UC Berkeley/Notebook/AL Protocols

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A. PCR (25ul) or wobble PCR (50ul)

I. PCR (25ul)

1. Mastermix: 20.375ul water, 0.5ul 10mM dNTP, 2.5ul 10x Buffer 2, 0.375ul Expand Polymerase 1.

a. Aliquot 23.75 ul.

2. Add 0.5ul Oligo 1 (10uM), 0.5ul Oligo 2 (10uM), 0.25 ul template DNA.

3. Load into thermocycler. **Small Holes are for 25ul and Big Holes for 50ul to maximize contact**

4. Select the program

II. Wobble PCR

1. Mastermix: 40ul water, 1.5 ul MgCl2, 5ul buffer (Taq), 1ul dNTP 10mM dNTP, 0.5 ul Taq Polymerase

a. Aliquot 48 ul

2. Add 1 ul 100 uM Oligo 1, 1 ul 100 uM Oligo 2.

3. Load and select wobble program

B. Use Clonewells to isolate DNA samples. Because sample B5 (K112312) may have been contaminated, PCR for b5 was done again.

C. Perform digestion using the follow procedure.

1. Make a solution of 5 ul NEB2, 15 ul DNA, 1 ul EcoRI, 1 ul BamHI, 28 ul water

2. Incubate for an hour

D. Clean up digestion with zymo columns.

1. Add 200ul ADB buffer to each of the digestion samples and pipette into zymo columns. Spin at 15s at full speed.

2. Add 200ul wash buffer. Spin for 15s at full speed.

3. Repeat step 2. Spin for an additional 90s at full speed.

4. Add 7ul of water to tube and spin for 30s at full speed.

E. Ligation

1. Mastermix: 6.5ul water, 1ul ligation buffer, 0.5 T4 DNA ligase, 1ul pBca1256. Aliquot 9ul.

2. Add 1ul of insert.

3. Cover with foil and incubate for 30 min at room temperature.

F. Transformation (always keep on ice)

1. 220ul competent cell in one tube, thaw on ice.

2. Add 30ul KCM and 20 ul water (both cold).

3. Invert ~2x to mix.

4. Aliquot 45ul cell into 10 ul of ligation rxn. (swirl and pipette up and down once)

5. Foil and Incubate 10 min on ice.

6. Heat shock 90s at 42 C.

7. Add 50 ul of LB.

8. Incubate 1 hr at 37 C.

9. Plate