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| Confirmation of DNA/RNA synthesis, agarose gels
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| (Old Title: Check DNA or RNA by 4.0% agarose gel Electrophoresis)
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| | | *[[/Plasmids|Swartz Lab Plasmid Database]] |
| Das lab, Stanford Biochemistry
| | *[[Agarose_gel_electrophoresis]] |
| For more information on protocols, see Das Lab Protocols
| | *[[PCR]] |
| First put together June 11, 2009, Rhiju Das.
| | *[[Assembly_pcr]] |
| Revised by Parin Sripakdeevong & Martin Claassen, May 02, 2011.
| | *[[Restriction_Digest]] |
| | | *[[DNA_ligation]] |
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| | *[[Transforming_chemically_competent_cells|Transform]] |
| Time estimate: ~1 hr 30 min
| | *[[Endy:Pouring_plates|Pouring Plates]] |
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| | *[[Maxiprep]] |
| A) For DNA: 4% agarose in 1X-TBE in EtBr
| | *[[Swartz: E.coli Transformation | E.coli Transformation]] |
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| A.1) Prepare 1X-TBE buffer (20 ml of 10x-TBE and adjust final volume to be 200 ml). Then add 10 ul of 10 mg/ml of Ethidium bromide (EtBr , 4 °C Fridge or room temp). Always make buffer fresh before run, because EtBr bleaches when exposed with light.
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| Note: EtBr bleaches when exposed to light. EtBr is also carcinogen and always use gloves when handling gel mix and buffer.
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| If fresh 10 x-TBE is required (usually already available in chemical shelf):
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| Tris-Base
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| 242.0 g
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| Boric acid
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| 102.7 g
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| Na2-EDTA salt
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| 7.44 g
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| *Nanopure H2O | |
| Adjusted to be 2 Litre
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| Autoclave the solution and keep at room temperature.
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| *Nanopure H2O at room B448, Nanopure is water from Water purification, Barnsted brand. Use water which has conductivity more than 14 megohm.
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| A.2) Weigh 3.0 g of agarose (Genepure E3120), add the agarose into 1 Liter-Bottle. Then add 75 ml of 1X-TBE buffer (containing EtBr) and heat agarose by microwave for 1 min and 45 sec, until agarose melt down.
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| Note: After microwave finished, continue to leave the gel solution in the microwave for a few minutes -- cool down to ~ 60 °C.
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| A.3) Pour the gel using a comb that will form wells large enough to accommodate at least 20 µl. Allow gel to solidify at room temperature for 30 mins.
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| A.4) Aliquot 2.5 ul of sample and add 2.5 µl of dH2O, then add sample buffer (6X) 2 ul.
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| If fresh 6X Gel loading buffer is required:
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| - Dissolve 25 mg bromophenol blue and 25 mg xylene cyanol FF
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| - Add 3 ml glycerol.
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| - Add 1.2 ml of 0.5 M EDTA (final 60 mM EDTA)
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| - Make up volume to 10 ml with distilled water.
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| - Store at 4°C.
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| A.5) Load the samples into the gel lanes (7 ul per lane). Also include ladder lanes (7ul per lane) for comparisons to your DNA length:
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| - 10 bp - ladder (Fermentas, SM1313)
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| - 20 bp - ladder (Fermentas, SM1323).
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| A.6) Attach and turn on the power supply, electrophoresis at 15 Watt for 20 min.
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| A.7) Turn off the power supply, carry the gel (contaminated with EtBr) in a weighing bowl.
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| A.8) Visualize the gel on a gel documentation system (gel-doc), UV transilluminator at room B412 (AlphaInnotech model EC) use channel 2 (knob on the top of the machine) for EtBr.
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| A.9) Open the gel-doc door, put your gel and close the door.
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| A.10) Turn on the lamp.
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| A.11) Visualize the image of your gel by clicking the “acquire” button.
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| A.12) Zoom, adjust light, focus & contrast, save and print out your image.
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| Ladder
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| 10 20 |----------- Samples -----------|
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| bp bp
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| 4% agarose in 1X-TBE and 10 ul of 10 mg/ml - EtBr, run 15 Watts x 20 min.
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| A.13) Remove your gel from gel-doc, discard the gel and clean up the gel-doc with kimwipes.
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| END.
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| B) For RNA: 4% agarose in 1X-TAE, 37% Formaldehyde and in Syber Green II
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| B.1) Prepare 1X-TAE buffer (4 ml of 50x-TAE and adjust final volume to be 200 ml). Then add 25 ul of 10,000X-Syber Green II, which keep in -20 ºC). Always make buffer fresh before run, because Syber Green II bleaches when exposed with light.
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| If fresh 50 x-TAE is required:
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| Tris-Base
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| 242.0 g
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| Glacial acetic acid
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| 57.1 ml
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| EDTA salt
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| 19.0 g
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| * Nanopure H2O | |
| Adjusted to be 1 Litre
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| Final pH should be 8.0 or Adjust with NaOH
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| Filter the solution and keep at room temperature.
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| * Nanopure H2O at room B448, Nanopure is water from Water purification, Barnsted brand. Use the water which has conductivity more than 14 megohm. | |
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| B.2) Weigh 3.0 g of agarose (Genepure E3120), add the agarose into 1 Liter-Bottle. Then add 75 ml of 1X-TAE buffer (containing Syber Green II) and heat agarose by microwave for 1 min and 45 sec, until agarose melt down.
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| Note: After microwave finished, continue leave the gel solution in the microwave for a few minutes.
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| B.3) Keep agarose to cool down to ~ 60 °C. then add 2.25 ml of 37% Formaldehyde (into 75 ml of gel mix) and mix well.
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| WARNING: Formaldehyde is toxic through skin contact and inhalation of vapors. Manipulations involving formaldehyde should be done in a chemical fume hood.
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| B.4) Pour the gel using a comb that will form wells large enough to accommodate at least 20 ul. Allow gel to solidify at room temp for 35 min.
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| B.5) Aliquot 4 ul of sample and add 6 ul of sample buffer (sample buffer composed of 50 mM EDTA in 10 mM Tris-HCl, pH 8.0, 0.1% Xylene Cyanol in 90% Formamide).
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| B.6) Heat denature samples at 90°C for 5 min, keep it cool down at room temp for 30 sec before load the sample on the gel.
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| B.7) Load the samples into the gel lanes (8 ul per lane). Also include ladder lanes (8 ul per lane) for comparisons of your RNA length:
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| - RNA low range ladder (SM 1831)
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| B.8) Electrophoresis at 15 Watt for 30 min.
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| B.9) Turn off the power supply, carry the gel in weighing bowl.
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| B.10) Visualize the gel on a gel documentation system (gel-doc), UV transilluminator at room B412 (AlphaInnotech model) use channel 3 (knob on the top of the machine) for Syber Green.
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| B.11) Open the gel-doc door, put your gel and close the door.
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| B.12) Turn on the lamp.
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| B.13) Visualize the image of your gel by clicking “acquire” button.
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| B.14) Zoom, adjust contrast, save and print out your image.
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| SM- |---------- Samples 0 Hrs ----------| |---------- Samples 5 Hrs ----------|
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| 1831
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| ladder
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| 4% agarose in 1X-TAE and 2.4 ml of 37% Formaldehyde (into 75 ml of gel mix), 1X-Syber Green II
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| run 15 Watt x30 min.
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| B.15) Remove your gel from gel-doc, discard the gel and clean up the gel-doc with kimwipes.
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| END.
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