Team:Stony Brook/MaterialsMethods

Materials and Methods Glossary

• Transformation of E. coli
• RNA Extraction
• Reverse Transcriptase to get cDNA
• PCR
• Gel Electrophoresis
• Ligation
• Digestion
• Qiaprep Spin Miniprep Kit
• Phosphotase

Transformation of E. coli

1. Thaw competent cells on ice
2. Add 50uL competent cells to all control and experimental microcentrifuge tubes.
3. Add 1-5uL DNA
4. Ice for 30 minutes
5. Heat shock in water bath at 42 C for 60 seconds
6. Ice for 5 minutes
7. Add 200uL SOC to each microcentrifuge tube
8. Incubate shaking at 37 C for 2 hours
9. Plate 50uL on appropriate media
10. Incubate plates at 37 C overnight

RNA Extraction

1. Pipet out 300 ul QIAzol lysis reagent
2. Use UVEX astrospec for eye protection and RNaseZap spray to sterilize area
3. Cut out tissue and place in reagent using a sterilized blade
4. Grind tissue in reagent in a down and twisting motion- until it looks relatively mashed
5. Vortex for a few seconds- until contents looked uniformly mixed (start and stop motion)
6. Let sit for 5 minutes at room temperature
7. 140 ul chloroform into reagent (do this quickly since chloroform expands)
8. Shake for 15 seconds (violently) - until "nice and gooey"
9. Let sit at room temperature for 2-3 minutes
10. Centrifuge at 12,000 rpm, 4 degrees celsius for 15 minutes
11. Pipet out the aqueous phase (top layer)
12. Add 525 ul ethanol to aqueous phase (pipet ethanol-aq phase up and down several times)
13. Pipet out 500 ul into column
14. Spin for 18 seconds at 10,000 rpm
15. Add another 500 ul
16. Spin for 18 seconds at 10,000 rpm
17. Add 350 ul buffer RWT solution
18. Centrifuge for 15 seconds at 10,000 rpm
19. Vortex solution
20. Take 80 ul - get close to white membrane but don't poke a hole in it
21. Let sit at room temperature for 15 minutes
22. Centrifuge for 18 seconds at room temperature (24 deg celsius) at 10,000 rpm
23. Pull column out of tube and dump the solution
24. Add 500 ul of RPE Buffer
25. Centrifuge for 18 seconds at 10,000 rpm - room temperature, dump column and centrifuge again for 1 minute
26. Retube it
27. Add 23 ul of water- pipet it right into the middle of the column membrane
28. Centrifuge again for 1 minute - product at bottom of second tube
29. Pipet out product
30. Centrifuge with hinge down for 1 min at room temperature
31. Put on ice

Reverse Transcriptase to get cDNA

1. In a sterile microfuge tube add:
   • RNA solution 1ng-1 µg total RNA or 1-100 ng polyA-selected RNA
   • Primer (50uM dT or 60uM Random Primer Mix) 2 µL 10mM dNTP mix 1 µL
   • nuclease-free H2O to final volume of 12 µL
2. (optional) Heat for 3-5 minutes at 65-70°C. Spin briefly and place promptly on ice.
3. Add:
   • 5X ProtoScript II RT Reaction bufferr 4 µL
   • 0.1M DTT 2ul
   • murine RNAse inhibitor (40U/ul) 1 µL
   • ProtoScript II Reverse Transcriptase (200U/ul) 1 µL
   So that the final volume is at 20 µL
4. Incubate at 42C for 30min-1hr. If Random Primer Mix is used, an incubation step at 25°C for 5 minutes is recommended before the 42°C incubation.
5. Inactivate enzyme at 80°C for 5 minutes.
6. Store products at -20°C or proceed to next step(s)

PCR

1. Assemble all reaction components on ice and quickly transfer the reaction components on ice and quickly transfer the reactions to a thermocycler preheated to the denaturation temperature (98 C). All components should be mixed prior to use.



Component	25 uL Reaction	50 uL Reaction	Final Concentration
Q5 High Fidelity 2X Master Mix	12.5 uL	50 ul Reaction	1X
10uM Forward Primer	1.25 uL	2.5 ul Reaction	0.5uM
10uM Reverse Primer	1.25 uL	2.5 ul Reaction	0.5uM
Template DNA	variable	variable	<1000ng
Nuclease Free Water	to 25 uL	to 25 uL	-



2. Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary
3. Transfer PCR tubes to a PCR machine and begin thermocycling. Begin initial denaturation at 98 C for 30 seconds. Then, for 25-35 cycles:
   • Run for 5-10 seconds at 98 C
   • Run for 10-30 seconds at a temperature between 50-72 degrees C. (The exact temperature is determined by the primers used).
   • Run for 20-30 seconds per kb at 72 C.
   • Run for 2 minutes at 72 C.
   • Pause the PCR at 4 C

Gel Electrophoresis

Creating the Agarose Gel:

1. Measure out 1g of agarose
2. Pour agarose power into microwaveable flask along with 100mL of 1X TAE
3. Microwave until the agarose is completely dissolved and there is a ncie roiling boil (1-3 minutes).
4. Let the agarose solution cool down for 5 minutes.
5. Pour the agarose into a gel tray with the well comb in place.
6. Place the newly poured gel at 4 C for 10-15 minutes or let sit at room temperature until it has completely solidified.

1. Add 2.5 uL loading dye to 7.5 uL of each sample.
2. Once solidified, place the agarose gel into the gel box.
3. Fill the gel box with 1xTAE until the gel is covered.
4. Carefully load a molecular weight ladder (8 uL) into one of the lanes.
5. Carefully load your samples into the additional wells of the gel (10uL of each sample)
6. Run the gel at 120 V until the dye line is approximately 75-80% of the way down the gel
7. Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.
8. Using UV light box, visualize DNA fragments

Ligation

1. Set up the following reaction in a microcentrifuge tube on ice. (T4 Ligase should be added last).
   
   

   Component	25 uL Reaction
   10X T4 DNA Ligase Buffer	2 uL
   Vector DNA	Variable (based on concentration)
   insert DNA	Variable (based on concentration)
   Nuclease-free Water	to 20uL
   T4 DNA Ligase	1uL



2. Gently mix the reaction by pipetting up and down and microcentrifuge briefly
3. For cohesive ends, incubate at room temperature for one hour.
4. Chill on ice. The ligation is ready for transformation.

Digestion

1. Spin down enzymes before use.
   
   

   Component	30 uL Reaction
   DNA	>2 ug
   Enzyme	1 uL
   Enzyme	1 uL
   Buffer	3 uL
   Water	To 30uL

   
   

Qiaprep Spin Miniprep Kit

Notes before starting:

• Add lyseblue reagent to Buffer P1 at a ratop pf 1:1000.
• Add the provided RNAse A solution to Buffer P1,mix, and store at 2-8 C
• Add ethanol (96-100&#37) to Buffer PE before use.
• All centrifugation steps are arried out at 13,000 rpm in a conventional tabletop microcentrifudge.

1. Pellet 1-5 mL bacterial overnight culture by centrifugation at >8000 rpm for 3 mins at room remperature (15-25 C)
2. Resuspend pelleted bacterial cells in 250 uL Buffer P1 and transfer to a microcentrifuge tube
3. Add 250 uL 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 minutes. If using Lyseblue reagent, the solution will turn blue.
4. Add 350 uL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times. If using lyseblue reagent, the solution will turn colorless.
5. Centrifuge for 10 minutes 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 30-60s and discard flow-through.
7. Wash the QiAlprep spin column by adding 500 uL Buffer PB. Centrifuge for 30-60 s and discard flow-through
8. Wash the QiAprep spin column by adding 750 uL Buffer PE. Centrifuge for 30-60s and discard the flowthrough. 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 centrifuge tube. To elute DNA, add 50 uL Buffer EB or water to the center of the QiAprep spin column, let it stand for 1 min, and centrifuge for 1 min

Phosphotase

1. Add 0.10 volume of 10X Antarctic Reaction Buffer to 1-5 ug of DNA
2. Add 1 uL of phosphatase and mix
3. Incubate for 60 minutes at 37 C
4. Heat-inactivte enzymes for 20 minutes at 80 C