Haynes Lab:Notebook/Engineering PC-TFs/2013/01/29: Difference between revisions
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==Summary== | ==Summary== | ||
* | |- valign="top" | ||
| <br><font size=3>'''Digestion/ ligation reaction'''</font> | |||
| <br>'''Dilute the purified PCR product to 20 fmol/μL''' | |||
* Measure ng/μL of the purified sample. | |||
* The volume of purified DNA (x) you will need to dilute in a final volume of 20 μL = 20 μL final volume * 20 fmols/μL * '''length in bp''' * 650 fg/fmol ÷ 1,000,000 fg/ng ÷ '''measured ng/μL''' | |||
** Formula: x = '''length in bp''' ÷ '''measured ng/μL''' * 0.26 | |||
|- | |||
| [[Image:Haynes_TIIS_fig7.png|250px|Figure 7]] | |||
| '''Perform BsmBI/ T4 ligase mediated assembly''' | |||
* BsmBI cuts the DNA fragments and creates complementary overhangs. | |||
* Complementary sticky ends anneal via base pairing. | |||
* T4 ligase seals gaps in the phosphodiester DNA backbone. | |||
{| {{table}} | |||
|- | |||
| bgcolor="grey" | Reagent | |||
| bgcolor="grey" | Vol. | |||
| rowspan=7 | '''Thermal cycling''' | |||
* [45°C, 2 min.; 16°C 5 min.] x25 | |||
* 60°C, 10 min. | |||
* 80°C, 20 min. | |||
* 4°C, ∞ | |||
|- | |||
| 20 fmol of each DNA part || up to 8.0 | |||
|- | |||
| 10x T4 ligase buffer (Promega) || 1.0 | |||
|- | |||
| T4 ligase (NEB) || 0.25 | |||
|- | |||
| BsmBI || 0.5 | |||
|- | |||
| dH<sub>2</sub>O || 0.25 | |||
|- | |||
| || 10.0 μL | |||
|} | |||
|} | |||
Revision as of 00:01, 1 February 2013
Engineering PC-TFs | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | |||||||||||||||
Summary | ||||||||||||||||
Digestion/ ligation reaction |
Dilute the purified PCR product to 20 fmol/μL
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Perform BsmBI/ T4 ligase mediated assembly
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