Team:RHIT/Protocol
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Protocols
- QIAprep Spin Miniprep Kit
- Pellet 1-5 ml bacterial overnight culture by centrifugation at >8000 rpm for 3 min at room temperature.
- Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube.
- Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min.
- Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
- Centrifuge for 10 min at 13,000 rpm in a table-top microcentrifuge.
- Apply the supernatant from step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 30-60 s and discard the flow-through.
- Recommended: Wash the QIAprep spin column by adding 500 µl Buffer PB. Centrifuge for 30-60 x and discard the flow-through.
- Wash the QIAprep spin column by adding 750 µl Buffer PE. Centrifuge for 30-60 s and discard the flow-through. Transfer the QIAprep spin column to the collection tube.
- Centrifuge for 1 min to remove residual wash buffer.
- Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 µl Buffer EB or water to the center of the QIAprep spin column, let it stand for 1 min, and centrifuge for 1 min.
- NEBuilder HiFi DNA Assembly Reaction Protocol
- Set up the following reaction on ice (for 2-3 fragment assembly):
- 0.03 - 0.2 pmols Total DNA (recommended DNA ratio - vector:insert = 1:2) X µl
- 10 µl NEBuilder HiFi DNA Assembly Master Mix
- 10 - X µl deionized water
- Incubate samples in a thermocycler at 50ºC for 15 minutes (when 2 or 3 fragments are being assembled) or 60 minutes (when 4-6 fragments are being assembled). Following incubation, store samples on ice or at -20ºC for subsequent transformation.
Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases. - Transform NEB 5-alpha Competent E. coli cells with 2 µl of the assembled product.
- Boiling Prep Protocol
- Spin down cells.
- Resuspend cells in 350 µl STET buffer.
- Add 25 µl of 10 mg/ml lysozyme, vortex.
- Boil 30-40 seconds in 100ºC water bath or 95ºC heat block.
- Add 10 µl of 10 mg/ml RNase.
- Centrifuge max speed 15 min.
- Add supernatant to 400 µl isopropanol. Invert to mix.
- Pellet 10 min. in centrifuge at max speed.
- Wash pellet with 70% ethanol.
- Resuspend in water or TE.
- NEB High Efficiency Transformation Protocol
- Thaw a tube of NE 5-alpha Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.
- Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
- Place the mixture on ice for 30 min. Do not mix.
- Heat shock at exactly 42ºC for exactly 30 seconds. Do not mix.
- Place on ice for 5 minutes. Do not mix
- Pipette 950 µl of room temperature SOC into the mixture.
- Place at 37ºC for 60 minutes. Shake vigorously (250 rpm) or rotate.
- Warm selection plates to 37ºC.
- Spread 50-100 µl of transformation onto a selection plate and incubate overnight at 37ºC. Alternatively, incubate at 30ºC for 24-36 hours or 25ºC for 48 hours.
- QIAquick Gel Extraction Kit
- Ethanol Precipitation
- Yeast Transformation (From Breeden Lab)