"
Team:SDU-Denmark/labwork/Protocols/3A
From 2012.igem.org
(Difference between revisions)
| Line 286: | Line 286: | ||
</br> | </br> | ||
<b>if sample comes from miniprep</b></br> | <b>if sample comes from miniprep</b></br> | ||
| - | + | Enzyme Master Mix for Plasmid Backbone (25ul total) | |
| - | + | ||
<ul type="square"> | <ul type="square"> | ||
| Line 309: | Line 308: | ||
<li>19 ul dH20 (0,5 ul less when using BSA</li> | <li>19 ul dH20 (0,5 ul less when using BSA</li> | ||
</ul> | </ul> | ||
| + | Digest Plasmid Backbone | ||
| + | <ul> | ||
| + | <li>Add 4 ul linearized plasmid backbone (25ng/ul for 100ng tota</li> | ||
| + | <li>Add 4 ul of Enzyme Master Mix</li> | ||
| + | <li>Digest 37C/30 min, heat kill Restriction Enzyme 80C/20 mi</li> | ||
</ol> | </ol> | ||
| - | <li></li> | + | <li>Digest step for Insert X:</li> |
Revision as of 22:04, 15 September 2012
| mRNA Isolation | PCR | Miniprep | Check Digest |
| 3A-Assembly | Content | Content | Content |
| Content | Content | Content | Content |
3A-Assembly -Ligation of 2 parts into a vector
- PCR step; see PCR protocol
- Digestion BSA is only needed for conventional restriction enzymes, NOT fast digest
- Digest step for Plasmid Backbone if sample comes from miniprep Enzyme Master Mix for Plasmid Backbone (25ul total)
- 5 ul Fastdigest greenbuffer / NEB Buffer 2
- 0.5 ul BSA
- 0.5 ul EcoRI-H
- 0.5 ul PstI
- 19 ul dH20 (0,5 ul less when using BSA)
- 5 ul Fastdigest greenbuffer / NEB Buffer
- 0.5 ul BSA
- 0.5 ul EcoRI-HF
- 0.5 ul PstI
- 0.5 ul DpnI (Used to digest any template DNA from productio
- 19 ul dH20 (0,5 ul less when using BSA
- Add 4 ul linearized plasmid backbone (25ng/ul for 100ng tota
- Add 4 ul of Enzyme Master Mix
- Digest 37C/30 min, heat kill Restriction Enzyme 80C/20 mi
- Digest step for Insert X:
