Difference between revisions of "HPP Assembly Procedure"
(Created page with ''''Edge Assembly''' This assembly uses BsmBI to free up the Half Edge Word in the first and second Half Edges. Ligase drives the reaction toward completion. Equimolar amounts …') |
|||
| (3 intermediate revisions by the same user not shown) | |||
| Line 1: | Line 1: | ||
'''Edge Assembly''' | '''Edge Assembly''' | ||
| − | This assembly uses BsmBI to free up the Half Edge Word in the first and second Half Edges. Ligase drives the reaction toward completion. Equimolar amounts of first and second Half Edges are used. Reactions are scaled up 5 fold to provide enough product in 5 ul for use with 10 ng of destination vector. | + | This assembly uses BsmBI to free up the Half Edge Word in the first and second Half Edges. Ligase drives the reaction toward completion. Equimolar amounts of first and second Half Edges are used. The Half Edges should be in a vector that has a different antibiotic resistance than the destination vector. Reactions are scaled up 5 fold to provide enough product in 5 ul for use with 10 ng of destination vector. |
| − | + | <br> | |
To determine the 5X amount for use in this reaction, use this formula: | To determine the 5X amount for use in this reaction, use this formula: | ||
| Line 9: | Line 9: | ||
10 ng destination vector x (Half Edge plasmid size/2114 bp destination size) x (6) | 10 ng destination vector x (Half Edge plasmid size/2114 bp destination size) x (6) | ||
| − | + | <br> | |
9 ul dH2O | 9 ul dH2O | ||
| Line 20: | Line 20: | ||
Total 25 ul | Total 25 ul | ||
| − | + | <br> | |
50 cycles of 50C for 2 minutes/16C for 3 minutes | 50 cycles of 50C for 2 minutes/16C for 3 minutes | ||
80C for 20 minutes | 80C for 20 minutes | ||
| − | + | <br> | |
Use 5ul of this Edge Assembly for Graph Assembly | Use 5ul of this Edge Assembly for Graph Assembly | ||
| + | |||
| + | '''Graph Assembly''' | ||
| + | |||
| + | This assembly uses BsaI to free up the Gene/Start/Finish Specific Words in the Graph Edges. Ligase drives the reaction toward completion. 5 ul of each Edge assembly is used. The destination vector should have an antibiotic resistance that is different from that of the Half Edge plasmids. | ||
| + | |||
| + | <br> | ||
| + | |||
| + | |||
| + | |||
| + | 5 ul Each Edge Assembly (1X Amount) | ||
| + | 6 ul dH2O | ||
| + | 1 ul (10 ng) Destination vector | ||
| + | 1 ul 10X Promega Ligase Buffer | ||
| + | 1 ul 500 mM NaCl | ||
| + | 0.5 ul BsaI | ||
| + | 0.5 ul T4 DNA Ligase | ||
| + | Total 10 ul + 5 x number of Edges | ||
| + | |||
| + | <br> | ||
| + | |||
| + | 50 cycles of 37C for 2 minutes/16C for 3 minutes | ||
| + | 80C for 20 minutes | ||
| + | |||
| + | <br> | ||
| + | |||
| + | Use 10 ul of this Graph Assembly for Transformation | ||
Latest revision as of 16:04, 30 August 2011
Edge Assembly
This assembly uses BsmBI to free up the Half Edge Word in the first and second Half Edges. Ligase drives the reaction toward completion. Equimolar amounts of first and second Half Edges are used. The Half Edges should be in a vector that has a different antibiotic resistance than the destination vector. Reactions are scaled up 5 fold to provide enough product in 5 ul for use with 10 ng of destination vector.
To determine the 5X amount for use in this reaction, use this formula:
10 ng destination vector x (Half Edge plasmid size/2114 bp destination size) x (6)
9 ul dH2O 5 ul First Half Edge (5X Amount) 5 ul Second Half Edge (5X Amount) 2.5 ul 10X Promega Ligase Buffer 2.5 ul 500 mM NaCl 0.5 ul BsmBI 0.5 ul T4 DNA Ligase Total 25 ul
50 cycles of 50C for 2 minutes/16C for 3 minutes 80C for 20 minutes
Use 5ul of this Edge Assembly for Graph Assembly
Graph Assembly
This assembly uses BsaI to free up the Gene/Start/Finish Specific Words in the Graph Edges. Ligase drives the reaction toward completion. 5 ul of each Edge assembly is used. The destination vector should have an antibiotic resistance that is different from that of the Half Edge plasmids.
5 ul Each Edge Assembly (1X Amount) 6 ul dH2O 1 ul (10 ng) Destination vector 1 ul 10X Promega Ligase Buffer 1 ul 500 mM NaCl 0.5 ul BsaI 0.5 ul T4 DNA Ligase Total 10 ul + 5 x number of Edges
50 cycles of 37C for 2 minutes/16C for 3 minutes 80C for 20 minutes
Use 10 ul of this Graph Assembly for Transformation
