McClean: Ladder Stock for PCR: Difference between revisions
(New page: Making 1KB Ladder Working Solution Working ladder solution is kept in the 4C fridge in the “Gels” box. If you use the last of it, please replace it for your coworkers! Stock is 1kb DN...) |
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==Overview== | |||
Working ladder solution is kept in the 4C fridge in the “Gels” box. If you use the last of it, please replace it for your coworkers! | Working ladder solution is kept in the 4C fridge in the “Gels” box. If you use the last of it, please replace it for your coworkers! | ||
Stock is 1kb DNA ladder NEB Cat #N3232L 500ug/ml concentration stored at -20C with the salmon sperm DNA. Working solution is 41.66 ug/ml in 10 mM Tris-Cl, pH 8.5 with loading buffer. | Stock is 1kb DNA ladder NEB Cat #N3232L 500ug/ml concentration stored at -20C with the salmon sperm DNA. Working solution is 41.66 ug/ml in 10 mM Tris-Cl, pH 8.5 with loading buffer. | ||
==Materials== | |||
# 1kb DNA ladder NEB Cat #N3232L 500ug/ml | |||
# 10mM Tris-Cl pH 8.5 (you can use EB Buffer from the Qiagen Kits!) | |||
# 6x Loading Dye Fisher Cat # BP633-5 | |||
==Procedure== | |||
To make a working solution mix 20ul of 500ug/ml ladder stock + 180ul of 10mM Tris-Cl pH 8.5 (you can use leftover EB buffer from the Qiagen kits!) + 40ul of 6x loading dye (Fisher Cat # BP633-5 located in the small chemicals box on the chemical shelf) in a sterile eppendorf tube. Make sure the tube is labeled (1X 1Kb ladder), dated, and initialed, and put back in the “Gels” box. | To make a working solution mix 20ul of 500ug/ml ladder stock + 180ul of 10mM Tris-Cl pH 8.5 (you can use leftover EB buffer from the Qiagen kits!) + 40ul of 6x loading dye (Fisher Cat # BP633-5 located in the small chemicals box on the chemical shelf) in a sterile eppendorf tube. Make sure the tube is labeled (1X 1Kb ladder), dated, and initialed, and put back in the “Gels” box. | ||
When running a gel, if you use 10ul of ladder, that will be ~0.4ug/lane. Use this along with the ladder datasheet (pasted to the front of the 4C fridge) to estimate the quantity of your DNA relative to the ladder (for example, the bright 3kb band contains ~100ng of DNA if you load 0.4ug per lane of the ladder). | When running a gel, if you use 10ul of ladder, that will be ~0.4ug/lane. Use this along with the ladder datasheet (pasted to the front of the 4C fridge) to estimate the quantity of your DNA relative to the ladder (for example, the bright 3kb band contains ~100ng of DNA if you load 0.4ug per lane of the ladder). | ||
==Notes== | |||
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! | |||
#List troubleshooting tips here. | |||
#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites. | |||
#Anecdotal observations that might be of use to others can also be posted here. | |||
Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip. | |||
==References== | |||
Adapted from: Kurt Thorn, 3/9/04 | |||
==Contact== | |||
*Who has experience with this protocol? | |||
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]]. | |||
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Revision as of 16:46, 13 July 2011
Overview
Working ladder solution is kept in the 4C fridge in the “Gels” box. If you use the last of it, please replace it for your coworkers! Stock is 1kb DNA ladder NEB Cat #N3232L 500ug/ml concentration stored at -20C with the salmon sperm DNA. Working solution is 41.66 ug/ml in 10 mM Tris-Cl, pH 8.5 with loading buffer.
Materials
- 1kb DNA ladder NEB Cat #N3232L 500ug/ml
- 10mM Tris-Cl pH 8.5 (you can use EB Buffer from the Qiagen Kits!)
- 6x Loading Dye Fisher Cat # BP633-5
Procedure
To make a working solution mix 20ul of 500ug/ml ladder stock + 180ul of 10mM Tris-Cl pH 8.5 (you can use leftover EB buffer from the Qiagen kits!) + 40ul of 6x loading dye (Fisher Cat # BP633-5 located in the small chemicals box on the chemical shelf) in a sterile eppendorf tube. Make sure the tube is labeled (1X 1Kb ladder), dated, and initialed, and put back in the “Gels” box. When running a gel, if you use 10ul of ladder, that will be ~0.4ug/lane. Use this along with the ladder datasheet (pasted to the front of the 4C fridge) to estimate the quantity of your DNA relative to the ladder (for example, the bright 3kb band contains ~100ng of DNA if you load 0.4ug per lane of the ladder).
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- List troubleshooting tips here.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- Anecdotal observations that might be of use to others can also be posted here.
Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
References
Adapted from: Kurt Thorn, 3/9/04
Contact
- Who has experience with this protocol?
or instead, discuss this protocol.
