Difference between revisions of "Diagnostic RP Digestion for Checking Insert Size"
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This procedure is for checking the insert size of a BioBrick part. It uses digestion with EcoRI + PstI. | This procedure is for checking the insert size of a BioBrick part. It uses digestion with EcoRI + PstI. | ||
| − | + | *Label a 1.5 ml tube for each of the digestions to be set up | |
| − | + | *Transfer about 300 ng into each tube and make up the difference with pure dH2O to 6 ul. Or just use 6 ul of a miniprep | |
| − | + | *If you have fewer than 4 digestions, add the following to each tube: | |
| − | + | 9 ul dH2O | |
| − | + | 2 ul 2 NEB4 10X buffer | |
| − | + | 2 ul 10X BSA | |
| − | + | 0.5 ul HF EcoRI | |
| − | + | 0.5 ul HF PstI | |
| − | + | *If you have 4 or more digestions, prepare a master mix as follows, where N is the number of digestions, then add 14 ul of it to each tube: | |
| − | + | 9 x (N+1) ul dH2O | |
| − | + | 2 x (N+1) ul 2 NEB4 10X buffer | |
| − | + | 2 x (N+1) ul 10X BSA | |
| − | + | 0.5 x (N+1) ul HF EcoRI | |
| − | + | 0.5 x (N+1) ul HF PstI | |
| − | + | *Spin down contents of each tube | |
| − | + | *Incubate at 37 C for 10 minutes | |
| − | + | *Add 5 ul blue sample buffer to each and run on gel | |
Latest revision as of 16:19, 21 June 2010
This procedure is for checking the insert size of a BioBrick part. It uses digestion with EcoRI + PstI.
- Label a 1.5 ml tube for each of the digestions to be set up
- Transfer about 300 ng into each tube and make up the difference with pure dH2O to 6 ul. Or just use 6 ul of a miniprep
- If you have fewer than 4 digestions, add the following to each tube:
9 ul dH2O 2 ul 2 NEB4 10X buffer 2 ul 10X BSA 0.5 ul HF EcoRI 0.5 ul HF PstI
- If you have 4 or more digestions, prepare a master mix as follows, where N is the number of digestions, then add 14 ul of it to each tube:
9 x (N+1) ul dH2O 2 x (N+1) ul 2 NEB4 10X buffer 2 x (N+1) ul 10X BSA 0.5 x (N+1) ul HF EcoRI 0.5 x (N+1) ul HF PstI
- Spin down contents of each tube
- Incubate at 37 C for 10 minutes
- Add 5 ul blue sample buffer to each and run on gel
