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Team:SDU-Denmark/Tools
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| - | To avoid having to manually calculate how many uL's of DNA and plasmid we should use to achieve optimal ligation circumstances, we created a small Program for the texas instruments calculators. It's simple called Ligation, and you can get it [[Team-SDU-Denmark-LIGATION.8xp.zip|here]]. | + | To avoid having to manually calculate how many uL's of DNA and plasmid we should use to achieve optimal ligation circumstances, we created a small Program for the texas instruments calculators. It's simple called Ligation, and you can get it [[Media:Team-SDU-Denmark-LIGATION.8xp.zip|here]]. |
=A plasmid editor ([http://www.biology.utah.edu/jorgensen/wayned/ape/ APE])= | =A plasmid editor ([http://www.biology.utah.edu/jorgensen/wayned/ape/ APE])= | ||
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| - | + | ==Ligation== | |
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| + | Q: How much DNA should be used in a ligation using T4 DNA Ligase? | ||
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| + | A: The unit definition uses 0.12 μM (300 μg/ml) lambda HindIII fragments. The high DNA concentration can be used for linker ligation. To promote circle formation, useful in transformation, a lower total DNA concentration should be used. The overall concentration of vector + insert should be between 1-10 μg/ml for efficient ligation. Insert:vector molar ratios between 2 and 6 are optimal for single insertions. Ratios below 2:1 result in lower ligation efficiency. Ratios above 6:1 promote multiple inserts. If you are unsure of your DNA concentrations, perform multiple ligations with varying ratios. From [http://www.neb.com/nebecomm/products/faqproductM0202.asp T4 DNA Ligase FAQ]. | ||
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Latest revision as of 00:02, 22 October 2009
Plasmid ligation helper
To avoid having to manually calculate how many uL's of DNA and plasmid we should use to achieve optimal ligation circumstances, we created a small Program for the texas instruments calculators. It's simple called Ligation, and you can get it here.
A plasmid editor ([http://www.biology.utah.edu/jorgensen/wayned/ape/ APE])
APE is a very powerful plasmid and linear DNA editor. It has helped us analyse our data from the very beginning. Among our favorite features are:
- Restriction site highlighting
- Expandable feature library with quick discovery. Perfect for analysing sequence results for biobrick inserts.
- DNA alignment feature. Another tool for analysing sequencing results
- Creates plasmid maps for easy reviewing
Ape was created by M. Wayne Davis. It runs on Windows, Linux and Mac OS X
Ligation
Q: How much DNA should be used in a ligation using T4 DNA Ligase?
A: The unit definition uses 0.12 μM (300 μg/ml) lambda HindIII fragments. The high DNA concentration can be used for linker ligation. To promote circle formation, useful in transformation, a lower total DNA concentration should be used. The overall concentration of vector + insert should be between 1-10 μg/ml for efficient ligation. Insert:vector molar ratios between 2 and 6 are optimal for single insertions. Ratios below 2:1 result in lower ligation efficiency. Ratios above 6:1 promote multiple inserts. If you are unsure of your DNA concentrations, perform multiple ligations with varying ratios. From [http://www.neb.com/nebecomm/products/faqproductM0202.asp T4 DNA Ligase FAQ].
