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Ligate DNA
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==Ligation Protocol== | ==Ligation Protocol== | ||
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| + | 1. Enter the names of the plasmids from which the vector and insert is obtained in the Excel worksheet. Also enter the lengths of the vector and insert fragments. This can be obtained by looking at the components section of the menu on the left hand side of the relevant plasmid display in Vector NTI. | ||
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| + | 2. The spreadsheet fills out the other columns to do a ligation with 100 ng of the vector fragment and a 1:3 molar ratio of vector to insert DNA. | ||
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| + | 3. All ligations are typically done at RT for 2 hours, however difficult ligations or ligations with low concentration of vector or inserts (<10 ng/µl) can be done for 16 hours at 16C. | ||
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| + | 4. Please keep the buffer on ice at all times when out of -20C. Keep the enzyme in the benchtop coolers at all times when out of the -20C. | ||
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| + | '''Ligation Calculations''' | ||
Revision as of 00:01, 29 October 2008
Ligation Protocol
1. Enter the names of the plasmids from which the vector and insert is obtained in the Excel worksheet. Also enter the lengths of the vector and insert fragments. This can be obtained by looking at the components section of the menu on the left hand side of the relevant plasmid display in Vector NTI.
2. The spreadsheet fills out the other columns to do a ligation with 100 ng of the vector fragment and a 1:3 molar ratio of vector to insert DNA.
3. All ligations are typically done at RT for 2 hours, however difficult ligations or ligations with low concentration of vector or inserts (<10 ng/µl) can be done for 16 hours at 16C.
4. Please keep the buffer on ice at all times when out of -20C. Keep the enzyme in the benchtop coolers at all times when out of the -20C.
Ligation Calculations
