DNA Protocols

Rehydrating Registry DNA

1. Add ddH₂O (10 μL) to appropriate well (will turn orange)
2. Incubate at room temperature for 10 min
3. Transform cells with DNA (1 μL)

Bacterial Transformation

1. Thaw 100 μL of competent cells on ice right before use (do not thaw completely)
2. Add DNA (max 20 μL) to cells, flick to mix, and incubate on ice for 30 min
3. Heat shock 2 min at 42 °C
4. Incubate on ice 5 min
5. Add 250 μL LB medium to each tube
6. Incubate 30-60 min at 37 °C shaking (If selecting on Kan incubation time is minimum 1 h)
7. Spin down (14,000 rpm for 2 min) and remove all supernatant except ~100 μL
8. Resuspend, spread 50 μL on antibiotic plate
9. Grow overnight in incubator at 37°C

Plasmid Miniprep (E. coli)

Reagents:

• Resuspension (P1): 50 mM Tris HCl (pH 8), 10 mM EDTA, 100 μg/mL RNase A (keep on ice, 4 °C)
• Lysis (P2): 200 mM NaOH, 1% SDS
• Precipitation (P3): 3 M K Acetate (pH 5.5)

1. Transfer O/N to 2 mL tube and pellet culture (spin at 14,000 rpm for 2 mins). Dump supernatant, and repeat for the rest of O/N.
2. Discard supernatant, resuspend pellet in P1 (300 μL)
3. Quickly: Add P2 (300 μL), invert gently 2-3x. Add P3 (300 μL), invert gently 2-3x
4. Centrifuge at 14,000 rpm, 10 mins, room temp
5. Without disturbing pellet, transfer supernatant to clean 1.5mL tube. Add isopropanol (650 μL). Wait 10 mins, room temp
6. Spin at 14,000 rpm for 10 mins at 4°C
7. Discard supernatant, add cold 70% etOH (500 μL)
8. Centrifuge at 14,000 rpm for 5 mins at 4°C
9. Carefully discard supernatant, dry pellet upside down
10. Resuspend in sterile ddH₂O (~30 μL)

Plasmid Miniprep (GenElute Kit)

1. Pellet cells from O/N culture
2. Discard supernatant, and resuspend with Resuspension solution (200 μL)
3. Add Lysis Solution (300 μL), immediately invert 6-8x (gently) until mixture becomes clear. Do not allow reaction to exceed 5 mins
4. Add Neutralization Solution (350 μL), invert 4-6x (gently). Pellet in centrifuge at max, 10 mins. If supernatant contains a large amount of floating particles, respin
5. Meanwhile, prepare binding column. Insert column into a microcentrifuge tube and add Column Prep Solution (500 μL). Spin at 13,000 rpm for 1 min, Discard flow through
6. Transfer the clear lysate (from step 4) to the column, and spin at 13,000 rpm, 1 min. Discard flow through
7. Add Wash Solution (750 μL) to column. Spin at 13,000 rpm, 1 min. Discard flow through and spin again at max, 2 mins
8. Transfer column a fresh collection tube and add ddH2O (~50 μL). Let sit 2 mins. Spin at 13,000 rpm, 1 min. Sample now ready for nano-drop

Gel Extraction (EZNA by Promega)

• Binding Buffer (XP2)
• SPW Wash Buffer (with ethanol)
• Transilluminator

1. Excise desired DNA bands from agarose gel using a transilluminator for visualization
2. Place each band into a 1.5 ml centrifuge tube and add one volume of Binding Buffer (XP2). Assume the density of agarose gel to be 1 g/1ml
3. Heat tubes at 60 °C for 7 minutes or until the gel has fully dissolved. Vortex frequently
4. Transfer the solution into a filter column placed within a 2 ml centrifuge tube. Do not exceed 700uL.
5. Centrifuge at 14,000 RPM for 1 minute. Repeat until entire solution has been filtered.
6. Discard solution and add 700 uL of SPW Wash Buffer to each column.
7. Centrifuge at 14,00 RPM for 1 minute. Discard solution and centrifuge again to dry completely.
8. Transfer columns two new 2 ml centrifuge tubes. Soak columns with 30-50 uL of water for 1 minute before eluting with 14,000 RPM centrifugation.

Restriction Digest

• 1 μg DNA
• 5 μL of CutSmart Buffer
• 0.5 to 1 μL enzyme 1
• 0.5 to 1 μL enzyme 2
• ddH₂O to bring total volume to 50 μL

Antarctic Phosphatase Treatment

1. To Vector tube from restriction digest, add 10x Antarctic Phosphatase Buffer (5 μL), ddH₂O (9 μL), and Antarctice phosphatase (1 μL)
2. Place tube in 37 °C water bath, 30 min
3. Transfer to 82 °C heatblock, 20min

Ligation (Using KAPA Rapid Ligase Kit)

• 10 μL Ligase Buffer
• 50 ng Vector DNA (from digest)
• 1:3 to 1:5 [Vector:Insert] molar ratio of Insert DNA (from digest)
• 1 μL Enzyme (Rapid Ligase)
• ddH₂O to bring total volume to 20 μL