Difference between revisions of "Help:Protocols/Restriction Digest"
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| Line 47: | Line 47: | ||
<table class="aliquot"> | <table class="aliquot"> | ||
<tr class="aliquotheader"><td></td><td>Part A</td><td>Part B</td><td>linearized plasmid backbone</td></tr> | <tr class="aliquotheader"><td></td><td>Part A</td><td>Part B</td><td>linearized plasmid backbone</td></tr> | ||
| − | <tr><th>DNA</th><td>250ng</td><td>250ng</td><td>250ng</td></tr> | + | <tr><th>DNA</th><td>250ng</td><td>250ng</td><td>250ng (10ul @ 25ng/ul)</td></tr> |
| + | <tr><th>dH2O</th><td>adjust to 16ul</td><td>adjust to 16ul</td><td>6ul</td></tr> | ||
<tr><th>NEB Buffer 2</th><td>2.5ul</td><td>2.5ul</td><td>2.5ul</td></tr> | <tr><th>NEB Buffer 2</th><td>2.5ul</td><td>2.5ul</td><td>2.5ul</td></tr> | ||
| − | <tr><th>BSA</th><td>0. | + | <tr><th>BSA</th><td>0.5ul</td><td>0.5ul</td><td>0.5ul</td></tr> |
<tr><th>Enzyme 1</th><td>0.5ul EcoRI</td><td>0.5ul XbaI</td><td>0.5ul EcoRI</td></tr> | <tr><th>Enzyme 1</th><td>0.5ul EcoRI</td><td>0.5ul XbaI</td><td>0.5ul EcoRI</td></tr> | ||
<tr><th>Enzyme 2</th><td>0.5ul SpeI</td><td>0.5ul PstI</td><td>0.5ul Pst1</td></tr> | <tr><th>Enzyme 2</th><td>0.5ul SpeI</td><td>0.5ul PstI</td><td>0.5ul Pst1</td></tr> | ||
Revision as of 13:20, 18 May 2012
| Template:HelpPage/MiniMap |
Restriction Digests |
| When using parts for 3A assembly, or testing the quality of a part you'll need to run a restriction digest. |
At iGEM HQ we recommend using the following protocols. The large reaction protocol will have enough of a volume for ligation and you'll be able to use a portion to run a gel. The small reaction protocol will only have enough volume for
Materials
- PCR tube
- dH20
- Enzymes (EcoRI, XbaI, SpeI, PstI)
- BSA
- Enzyme Buffer (NEBuffer 2)*
Notes: You should keep all materials on ice.
Procedure
- Add 250ng of DNA to be digested, and adjust with dH20 for a total volume of 16ul.
- Add 2.5ul of NEBuffer 2 to the tube.
- Add 0.5ul of BSA to the tube.
- Add 0.5ul of your first enzyme.
- Add 0.5ul of your second enzyme.
- There should be a total volume of 20ul. Mix well and spin down briefly.
- Incubate the restriction digest at 37C for 30min, and then 80C for 20min to heat kill the enzymes.
We incubate in a thermal cycler with a heated lid - Run a portion of the digest on a gel, to check that both plasmid and part length are accurate. Use ~2ul of the digest (20ng of DNA) for ligations.
3A Assembly Procedure
| Part A | Part B | linearized plasmid backbone | |
| DNA | 250ng | 250ng | 250ng (10ul @ 25ng/ul) |
|---|---|---|---|
| dH2O | adjust to 16ul | adjust to 16ul | 6ul |
| NEB Buffer 2 | 2.5ul | 2.5ul | 2.5ul |
| BSA | 0.5ul | 0.5ul | 0.5ul |
| Enzyme 1 | 0.5ul EcoRI | 0.5ul XbaI | 0.5ul EcoRI |
| Enzyme 2 | 0.5ul SpeI | 0.5ul PstI | 0.5ul Pst1 |