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.

Reference: Qiagen; Netherlands; Product #27104



• 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.

Reference: New England Biolabs; Ipswich, MA; Product #E5520S



• 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.

Reference: Sambrook, J., & Russell, D. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.



• 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.

Reference: New England Biolabs; Ipswich, MA



• QIAquick Gel Extraction Kit
1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
2. Weigh the gel slice in a colorless tube. Add 3 volumes Buffer QG to 1 volume gel (100 mg ~ 100 µl).
3. Incubate at 50ºC for 10 min (or until the gel slice has completely dissolved). Vortex the tube every 2-3 min to help dissolve gel.
4. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 µl 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow.
5. Add 1 gel volume of isopropanol to the sample and mix.
6. Place a QIAquick spin column in a provided 2 ml collection tube.
7. To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min. Discard flow-through and place the Qiaquick column back into the same tube. For sample volumes of >800 µl, load and spin/apply vacuum again.
8. If the DNA will subsequently be used for sequencing, in vitro transcription, or microinjection, add 0.5 ml Buffer QG to the QIAquick column and centrifuge for 1 min. Discard flow-through and place the QIAquick column back into the same tube.
9. To wash, add 0.75 ml Buffer PE to QIAquick column and centrifuge for 1 min. Discard flow-through and place the QIAquick column back into the same tube.
10. Centrifuge the QIAquick column once more in the provided 2 ml collection tube for 1 min at 13,000 rpm to remove residual wash buffer.
11. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
12. To elute DNA, add 50 µl Buffer EB or water to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 µl Buffer EB to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min. After the addition of Buffer EB to the QIAquick membrane, increasing the incubation time to up to 4 min can increase the yield of purified DNA.
13. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.

Reference: Qiagen; Netherlands; Product #28704



• Ethanol Precipitation
1. Estimate the volume of the DNA solution.
2. Adjust the concentration of monovalent cations either by dilution with TE (pH 8.0) if the DNA solution contains a high concentration of salts of by addition of one of the salt solutions shown in Table A8-1. (If the volume of the final solution is 400 µl or less, carry out precipitation in a single microcentrifuge tube. Larger volumes can be divided among several microfuge tubes, or the DNA can be precipitated and centrifuged in tubes that will fit in a medium-speed centrifuge or ultracentrifuge.)
3. Mix the solution well. Add exactly 2 volumes of ice-cold ethanol and again mix the solution well. Store the ethanolic solution on ice to allow the precipitate of DNA to form. (Usually 15-30 minutes is sufficient, but when the size of the DNA is small (<100 nucleotides) or hen it is present in small amounts (<0.1 µg/ml), extend the period of storage to at least 1 hour and add MgCl2 to a final concentration of 0.01 M. DNA can be stored indefinitely in ethanolic solutions at 0ºC or at -20ºC.)
4. Recover the DNA by centrifugation at 0ºC. (For most purposes, centrifugation at maximum speed for 10 minutes in a microfuge is sufficient. However, as discussed above, when low concentrations of DNA (<20 ng/ml) or very small fragments are being processed, more extensive centrifugation may be required.)
5. Carefully remove the supernatant with an automatic micropipettor or with a disposable pipette tip attached to a vacuum line. Take care not to disturb the pellet of nucleic acid (which may be invisible). Use the pipette tip to remove any drops of fluid that adhere to the walls of the tube. (It is best to save the supernatant from valuable DNA samples until recovery of the precipitated DNA has been verified.)
6. Fill the tube halfway with 70% ethanol and recentrifuge at maximum speed for 2 minutes at 4ºC in a microfuge.
7. Repeat step 5.
8. Store the open tube on the bench at room temperature until the last traces of fluid have evaporated.
9. Dissolve DNA pellet in the desired volume of buffer (usually TE). Rinse the walls of the tube with the buffer.

Reference: Sambrook, J., & Russell, D. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.



• Yeast Transformation (From Breeden Lab)
1. Pick colonies and inoculate cultures in YPD and grow overnight.
2. Measure OD₆₀₀ nm and dilute to OD₆₀₀ nm = 0.3 in 50 ml.
3. Grow for 3 hours shaking at 30℃.
4. Separate into two 25 ml aliquots and spin at 3,000 rpm for 5 minutes. Wash cells with 12.5 ml H2O.
5. Spin (3,000 rpm 5 minutes)and resuspend in 1 ml 1 x LiAc/0.5 x TE.
6. Incubate at room temperature for 10 minutes.
7. Boil salmon sperm DNA for 5 minutes and place on ice. Vortex before use.
8. Combine in tube: 100 µl cells, 10 µl salmon sperm DNA (10 mg/ml), and 5 µl plasmid DNA.
9. Add 700 µl 1 x LiAc/1 x TE/40% PEG 3350 mix.
10. Incubate for 30 minutes shaking at 30℃.
11. Add 85 µl DMSO.
12. Heat shock at 42℃ for 7 minutes.
13. Spin and resuspend cells in 1 ml 1 x TE
14. Spin and resuspend cells in 0.5 ml 1 x TE.
15. Place 100 - 200 µl per plate and incubate at 30℃ for three days.

http://research.fhcrc.org/breeden/en/methods.html