Lidstrom:Loading Dye Recipe: Difference between revisions
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Amanda uses the following recipe that was once found online: | Amanda uses the following recipe that was once found online: | ||
* perhaps she saw [http://www.wendychao.com/science/protocols/recipes/ this link]. | |||
==10X Loading Buffer== | ==10X Loading Buffer== | ||
*Composition: 25% Ficoll, 100 mM Tris-HCL & EDTA, Xylene Cyanol, Orange G | *Composition: 25% Ficoll, 100 mM Tris-HCL & EDTA, Xylene Cyanol, Orange G | ||
Recipe: | Recipe: | ||
*2.5 g Ficoll-400 | *2.5 g [http://www.tdbcons.se/fitcficoll/ Ficoll-400] | ||
*1 mL 1M Tris-Cl, pH 7.4 | *1 mL 1M Tris-Cl, pH 7.4 | ||
** Tris-Cl = Tris-HCl | ** Tris-Cl = Tris-HCl | ||
| Line 20: | Line 21: | ||
10X Loading Buffer (50 mL batch) | 10X Loading Buffer (50 mL batch) | ||
*12.5 g Ficoll-400 | *12.5 g [http://www.tdbcons.se/fitcficoll/ Ficoll-400] | ||
*5 mL 1M Tris-Cl, pH 7.4 | *5 mL 1M Tris-Cl, pH 7.4 | ||
** Tris-Cl = Tris-HCl | ** Tris-Cl = Tris-HCl | ||
| Line 26: | Line 27: | ||
*Bring to 50 mL with ddH2O, heating to 65oC to dissolve | *Bring to 50 mL with ddH2O, heating to 65oC to dissolve | ||
*Add dye as you fancy. | *Add dye as you fancy. | ||
==Janet's Notes== | |||
* Janet made this 7/16/2012. I found 0.5 EDTA to be insoluble, and noted that a [http://www.wendychao.com/science/protocols/recipes/ reference] made this a pH 8 solution. I raised the pH with 10 N NaOH (found by the pH probe) and it increased solubility, but there was still a lot of crystalline EDTA. I figured the amount of EDTA added didn't matter because most recipes don't include it. Having it is just a bonus for DNA stability. Further, other recipes that do have it use a much lower concentration. | |||
* I added H2O to 50 mL and let the solution sit overnight to dissolve. The resulting solution had a volume of 45 mL and was less viscous than Amanda's. I added 1g more ficoll 400 & used a heater to help solubilize it, then left it on the desk overnight to finish the job. | |||
Revision as of 11:44, 16 July 2012
Back to Lidstrom:Protocols
First, see the OpenWetWare info.
Amanda uses the following recipe that was once found online:
- perhaps she saw this link.
10X Loading Buffer
- Composition: 25% Ficoll, 100 mM Tris-HCL & EDTA, Xylene Cyanol, Orange G
Recipe:
- 2.5 g Ficoll-400
- 1 mL 1M Tris-Cl, pH 7.4
- Tris-Cl = Tris-HCl
- 2 mL 0.5 M EDTA
- Add ddH20 to 10 mL, heating to 65oC to dissolve
- Add 25-50 mg of xylene cyanol and 25-50 mg Orange G.
If needed, adjust dye intensity with colorless 10x buffer. Store at room temperature.
Note: I recommend first making 50 mL 10X loading buffer WITHOUT any marker dyes. You can then add different combinations of dyes and dilute them as you wish to attain your preferred color intensity. This will yield enough loading buffer for your entire career.
10X Loading Buffer (50 mL batch)
- 12.5 g Ficoll-400
- 5 mL 1M Tris-Cl, pH 7.4
- Tris-Cl = Tris-HCl
- 10 mL 0.5 M EDTA
- Bring to 50 mL with ddH2O, heating to 65oC to dissolve
- Add dye as you fancy.
Janet's Notes
- Janet made this 7/16/2012. I found 0.5 EDTA to be insoluble, and noted that a reference made this a pH 8 solution. I raised the pH with 10 N NaOH (found by the pH probe) and it increased solubility, but there was still a lot of crystalline EDTA. I figured the amount of EDTA added didn't matter because most recipes don't include it. Having it is just a bonus for DNA stability. Further, other recipes that do have it use a much lower concentration.
- I added H2O to 50 mL and let the solution sit overnight to dissolve. The resulting solution had a volume of 45 mL and was less viscous than Amanda's. I added 1g more ficoll 400 & used a heater to help solubilize it, then left it on the desk overnight to finish the job.
