Biomod/2012/UT/Nanowranglers/Methods: Difference between revisions
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==Making/Running 12% Denaturing Gel== | ==Making/Running 12% Denaturing Gel== | ||
*Making gel: | *Making gel: | ||
:In a 50 mL conical tube, mix 30 mL of 20% Acrylamide in 1X TBE, 20 mL of 1X TBE Denaturing PAGE dilution buffer | |||
:Add 500 µL of 10% APS and 50 µL of TEMED, mix thoroughly by inverting | |||
:Immediately pour gel solution into glass plate assembly, apply a comb, wait for gel to solidify | |||
*Running gel: | *Running gel: | ||
:Prepare sample by mixing DNA sample with equal volume of 2X Denaturing Dye | |||
:Denature sample by heating at 90 °C for 5 minutes | |||
:Attach gel assembly to PAGE rig, fill top and bottom chambers with 1X TBE | |||
:Clear lanes of excess urea by pipetting | |||
:Pre-run gel at 450 V for 10-20 minutes | |||
:Load samples, run gel at 450 V with a fan | |||
==Making/Running 12% Native Gel== | ==Making/Running 12% Native Gel== |
Revision as of 11:02, 22 October 2012
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Making 3% Agarose Gel
- In a 250 mL glass container, mix 3 g of Metaphor agarose powder, 100 mL of 1X TBE buffer, then weigh
- Heat container in microwave on medium for 2 minutes
- Remove, swirl, continue heating on high until all particles are dissolved
- Reweigh solution in container, add hot distilled water to restore initial weight, swirl
- Cool solution to 60 °C, pour into a casting tray, apply a comb
- Place gel at 4 °C for 20 minutes, then at room temperature for 10 minutes
Making/Running 12% Denaturing Gel
- Making gel:
- In a 50 mL conical tube, mix 30 mL of 20% Acrylamide in 1X TBE, 20 mL of 1X TBE Denaturing PAGE dilution buffer
- Add 500 µL of 10% APS and 50 µL of TEMED, mix thoroughly by inverting
- Immediately pour gel solution into glass plate assembly, apply a comb, wait for gel to solidify
- Running gel:
- Prepare sample by mixing DNA sample with equal volume of 2X Denaturing Dye
- Denature sample by heating at 90 °C for 5 minutes
- Attach gel assembly to PAGE rig, fill top and bottom chambers with 1X TBE
- Clear lanes of excess urea by pipetting
- Pre-run gel at 450 V for 10-20 minutes
- Load samples, run gel at 450 V with a fan
Making/Running 12% Native Gel
- Making gel:
- In a 50 mL conical tube, mix 15 mL of 40% Acyrlamide, 5 mL of 100% Glycerol, 5 mL of autoclaved 10X TBE, 25 mL of sterilized deionized water (sd H2O)
- Add 500 µL of 10% APS and 50 µL of TEMED, mix thoroughly by inverting
- Immediately pour gel solution into glass plate assembly, apply a comb, wait for gel to solidify
- Running gel:
- Prepare sample by mixing DNA sample with 1/5 volume of 6X Orange Dye
- Attach gel assembly to PAGE rig, fill top and bottom chambers with 1X TBE
- Clear lanes of excess urea by pipetting
- Pre-run gel at 300 V for 10-20 minutes
- Load samples, run gel at 300 V with a fan
Visualizing/Excising Gel
- Visualizing gel:
- Add 5 µL of 10,000X SYBR Gold Nucleic Acid Gel Stain, 50 mL of 1X TBE to glass dish
- Remove gel from glass plates into dish, let incubate on rotator for 15 minutes
- Scan for Fluorescence Intensity of Storm Imager
- Excising gel:
- Print actual size image of gel
- Move gel on top of saran-wrapped PAGE glass plate, place over image
- Use new razor to cut DNA band, place into 1.7 mL tube
DNA Elution following Denaturing or Native PAGE
- Following Denaturing PAGE:
- Crush isolated DNA with plunger rod until gel is fine
- Suspend gel in 500 µL of 1X TBE
- Place tube on shaking incubator at 80 °C on high for 15 minutes
- Spin down at 14,000 rpm for 2 minutes, transfer supernatant to filter-based centrifuge tube
- Concentrate DNA by ethanol precipitating flow-through
- Following Native PAGE:
- Crush isolated DNA with plunger rod until gel is fine
- Suspend gel in 1 mL of 1X TBE
- Elute DNA overnight at 37 °C
- Spin down at 14,000 rpm for 2 minutes, transfer supernatant to filter-based centrifuge tube
- Add flow-through to concentrator filter until concentrated down to approximately 50 µL
Ethanol Precipitation
- Add 2.5 V of 100% ethanol, 0.1 V of 3 M NaAc (pH 5.2), 2 µL of glycogen to initial volume of solution
- Vortex, place in -80 °C freezer for 15 minutes
- Spin down at 13,000 rpm for 15 minutes at 4 °C
- Remove supernatant from pellet, wash pellet by adding 1 mL of 70% ethanol
- Spin down at 13,000 rpm for 5 minutes, discard supernatant
- Dry using speed vac for 10-20 minutes
- Resuspend DNA in sterilized deionized water
Forming/Checking Hairpins
- Denature strands by heating to 90 °C for 1 minute then slowly decreasing the temperature to 37 °C at 0.1 °C s-1
- Incubate strands at 37 °C
- Transfer 20 µL of each tube to new tubes
- Add 1/5 V of 6X Orange DNA Loading Dye to each tube
- Load 20 µL of each samples into 12% native gel
- Run gel at 300 V with a fan
- Visualize DNA
Walker System Assembly
- Substrate 1 (S1) Assembly:
- Combine hairpin 02aA, track strand 01Tb, hairpin 03bB
- Anneal by heating system at 95 °C for 5 minutes, then cooling to 37 °C at 0.24 °C min-1
- Walker (W) Assembly:
- Combine W1-BHQ1, W2-BHQ1
- Anneal by heating mixture to 95 °C for 5 minutes, then cooling to 37 °C at 0.24 °C min-1
- S1-W Assembly:
- Incubate Substrate 1 (S1) and Walker (W) at 37 °C for 3 hours
- Substrate 2 Assembly
- Combine hairpin 04cA, track strand 01Ta, hairpin 05dB-JOE, hairpin 06eA-TMR, 07f-FAM
- Anneal by heating system to 95 °C for 5 minutes, then cooling to 37 °C at 0.24 °C min-1
- Final System (S1-W-S2) Assembly:
- Incubate S2 and S1-W at 37 °C for 3 hours
Buffers Used
- 10X TBE:
- Tris base (pH 8.0, F.W. 121.1): 107.8 g (890 mM)
- EDTA (F.W. 372.2): 7.44 g (20 mM)
- Boric acid (F.W. 61.83): 55.03 g (890 mM)
- sd H2O: to 1 L
- Filtered with .0.2 uM
- 10 X TMgK:
- Tris (pH 8.0): 9.86 g Tris-HCl (F.W. 157.6) + 4.532 g Tris base (F.W. 121.1), adjust to 100 mL with sd H2O (1 M)
- MgCl2 6H2O (F.W. 203.3): 20.33 g, adjust to 100 mL with sd H2O (1 M)
- KCl (F.W. 74.55): 7.455 g, adjust to 100 mL with sd H2O (1 M)
- Tris (pH 8.0, 1 M): 10 mL (100 mM)
- MgCl2 (1 M): 4 mL (40 mM)
- KCl (1 M): 15 mL (150 mM)
- sd H2O: to 100 mL
- Filtered with .2 uM