Team:Calgary/Notebook/ProtocolManual/DNA
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Revision as of 22:34, 17 October 2014
DNA Protocols
Rehydrating Registry DNA
- Add ddH2O (10μL) to appropriate well (will turn orange)
- Incubate at room temperature for 10 min
- Transform cells with DNA (1μL)
Bacterial Transformation
- Thaw 100μL of competent cells on ice right before use (do not thaw completely)
- Add DNA (max 20μL) to cells, flick to mix, and incubate on ice for 30 min
- Heat shock 2 min at 42°C
- Incubate on ice 5 min
- Add 250μL LB medium to each tube
- Incubate 30-60 min at 37°C shaking (If selecting on Kan incubation time is minimum 1h)
- Spin down (14,000 rpm for 2 min) and remove all supernatant except ~100μL
- Resuspend, spread 50μL on antibiotic plate
- Grow overnight in incubator at 37°C
Plasmid Miniprep (E. coli)
Reagents:
- Resuspension (P1): 50mM Tris HCl (pH 8), 10mM EDTA, 100μg/mL RNase A (keep on ice, 4°C)
- Lysis (P2): 200mM NaOH, 1% SDS
- Precipitation (P3): 3M K Acetate (pH 5.5)
- Transfer O/N to 2mL tube and pellet culture (spin at 14,000 rpm for 2 mins). Dump supernatant, and repeat for the rest of O/N.
- Discard supernatant, resuspend pellet in P1 (300μL)
- Quickly: Add P2 (300 μL), invert gently 2-3x. Add P3 (300 μL), invert gently 2-3x
- Centrifuge at 14,000 rpm, 10mins, room temp
- Without disturbing pellet, transfer supernatant to clean 1.5mL tube. Add isopropanol (650 μL). Wait 10 mins, room temp
- Spin at 14,000 rpm for 10 mins at 4°C
- Discard supernatant, add cold 70% etOH (500 μL)
- Centrifuge at 14,000 rpm for 5 mins at 4°C
- Carefully discard supernatant, dry pellet upside down
- Resuspend in sterile ddH2O (~30 μL)
Plasmid Miniprep (GenElute Kit)
- Pellet cells from O/N culture
- Discard supernatant, and resuspend with Resuspension solution (200 μL)
- Add Lysis Solution (300 μL), immediately invert 6-8x (gently) until mixture becomes clear. Do not allow reaction to exceed 5 mins
- 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
- 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
- Transfer the clear lysate (from step 4) to the column, and spin at 13,000 rpm, 1 min. Discard flow through
- Add Wash Solution (750 μL) to column. Spin at 13,000 rpm, 1 min. Discard flow through and spin again at max, 2 mins
- 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
- Excise desired DNA bands from agarose gel using a transilluminator for visualization
- Place each band into a 1.5ml centrifuge tube and add one volume of Binding Buffer (XP2). Assume the density of agarose gel to be 1g/1ml
- Heat tubes at 60°C for 7 minutes or until the gel has fully dissolved. Vortex frequently
- Transfer the solution into a filter column placed within a 2ml centrifuge tube. Do not exceed 700uL.
- Centrifuge at 14,000RPM for 1 minute. Repeat until entire solution has been filtered.
- Discard solution and add 700uL of SPW Wash Buffer to each column.
- Centrifuge at 14,00RPM for 1 minute. Discard solution and centrifuge again to dry completely.
- Transfer columns two new 2ml centrifuge tubes. Soak columns with 30-50uL of water for 1 minute before eluting with 14,000RPM centrifugation.
Restriction Digest
Separately, into labeled tubes for Insert and Vector, add the following reagents:
- 1μg DNA
- 5μL of CutSmart Buffer
- 0.5 to 1μL enzyme 1
- 0.5 to 1μL enzyme 2
- ddH2O to bring total volume to 50μL
Antarctic Phosphatase Treatment
- To Vector tube from restriction digest, add 10x Antarctic Phosphatase Buffer (5 μL), ddH2O (9 μL), and Antarctice phosphatase (1 μL)
- Place tube in 37°C water bath, 30 min
- Transfer to 82°C heatblock, 20min
Ligation (Using KAPA Quick Ligase Kit)
- 10 μL Ligase Buffer
- 50ng Vector DNA (from digest)
- 1:3 to 1:5 [Vector:Insert] molar ratio of Insert DNA (from digest)
- 1 μL Enzyme (Quick Ligase)
- ddH2O to bring total volume to 20 μL