Difference between revisions of "Team:RHIT/Protocol"

Protocols

• QIAprep Spin Miniprep Kit
1. Pellet 1-5 ml bacterial overnight culture by centrifugation at >8000 rpm for 3 min at room temperature.
2. Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube.
3. 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.
4. Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
5. Centrifuge for 10 min at 13,000 rpm in a table-top microcentrifuge.
6. 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.
7. Recommended: Wash the QIAprep spin column by adding 500 µl Buffer PB. Centrifuge for 30-60 x and discard the flow-through.
8. 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.
9. Centrifuge for 1 min to remove residual wash buffer.
10. 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
1. 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
2. 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.
3. Transform NEB 5-alpha Competent E. coli cells with 2 µl of the assembled product.


• Boiling Prep Protocol
1. Spin down cells.
2. Resuspend cells in 350 µl STET buffer.
3. Add 25 µl of 10 mg/ml lysozyme, vortex.
4. Boil 30-40 seconds in 100ºC water bath or 95ºC heat block.
5. Add 10 µl of 10 mg/ml RNase.
6. Centrifuge max speed 15 min.
7. Add supernatant to 400 µl isopropanol. Invert to mix.
8. Pellet 10 min. in centrifuge at max speed.
9. Wash pellet with 70% ethanol.
10. Resuspend in water or TE.


• NEB High Efficiency Transformation Protocol
1. 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.
2. 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.
3. Place the mixture on ice for 30 min. Do not mix.
4. Heat shock at exactly 42ºC for exactly 30 seconds. Do not mix.
5. Place on ice for 5 minutes. Do not mix
6. Pipette 950 µl of room temperature SOC into the mixture.
7. Place at 37ºC for 60 minutes. Shake vigorously (250 rpm) or rotate.
8. Warm selection plates to 37ºC.
9. 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)