Haynes Lab:Notebook/Engineering PC-TFs/2013/01/29: Difference between revisions
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|style="background-color: # | |style="background-color: #FFCC00"|<br>[[Image:Hayneslab3.gif|200px|center]][[Image:Asu logo 3.gif|200px|center]]<span style="font-size:22px;"><br></span> | ||
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<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | ||
== | ==1/29/13== | ||
Type IIS Assembly Day 2 | |||
---- | |||
<br><font size=3>'''Digestion/ ligation reaction'''</font> | |||
* <br>'''Dilute the purified PCR product to 20 fmol/μL''' | * <br>'''Dilute the purified PCR product to 20 fmol/μL''' | ||
* Measure ng/μL of the purified sample. | * 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''' | * 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 | ** Formula: x = '''length in bp''' ÷ '''measured ng/μL''' * 0.26 | ||
<font size=3>'''Purified PCR Product to 20fmol/μL'''</font> | |||
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | |||
|- valign="top" | |||
| bgcolor=#cfcfcf | Sample | |||
| bgcolor=#cfcfcf | μL of Sample | |||
| bgcolor=#cfcfcf | μL of dH2O | |||
|- | |||
| 1. pSB1A3 || 7.8|| 12.2 | |||
|- | |||
| 2. hPCD || 2.8 || 17.2 | |||
|- | |||
| 3. BL02 || 6.4|| 13.6 | |||
|- | |||
| 4. BL03 || 5.5 || 14.5 | |||
|- | |||
| 5. BL04 || 8.8|| 11.2 | |||
|- | |||
| 6. fshPCD || 2.3|| 7.7 | |||
|} | |||
* '''Perform BsmBI/ T4 ligase mediated assembly''' | * '''Perform BsmBI/ T4 ligase mediated assembly''' | ||
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* T4 ligase seals gaps in the phosphodiester DNA backbone. | * T4 ligase seals gaps in the phosphodiester DNA backbone. | ||
{| {{table}} | {| {{table}} | ||
| bgcolor="grey" | Reagent | | bgcolor="grey" | Reagent | ||
| bgcolor="grey" | Vol. | | bgcolor="grey" | Vol. | ||
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* 4°C, ∞ | * 4°C, ∞ | ||
|- | |- | ||
| 20 fmol of each DNA part || up to 8.0 | | 20 fmol (1 μL) of each DNA part || up to 8.0 | ||
|- | |- | ||
| 10x T4 ligase buffer (Promega) || 1.0 | | 10x T4 ligase buffer (Promega) || 1.0 | ||
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|- | |- | ||
| || 10.0 μL | | || 10.0 μL | ||
|} | |||
{| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table --> | |||
|- valign="top" | |||
| bgcolor="grey" | Reagent | |||
| bgcolor="grey" | 1 | |||
| bgcolor="grey" | 2 | |||
| bgcolor="grey" | 3 | |||
|- | |||
| gg2 pSB1A3 || 1.0 || 1.0 || 1.0 | |||
|- | |||
| gg3 hPCD || 1.0 || 1.0 || 1.0 | |||
|- | |||
| BL02 || 1.0 || --- || --- | |||
|- | |||
| BL03 || --- || 1.0 || --- | |||
|- | |||
| BL04 || --- || --- || 1.0 | |||
|- | |||
| 10x ligase buffer || 1.0 || 1.0 || 1.0 | |||
|- | |||
| NEB T4 lgase || 0.25 || 0.25 || 1.0 | |||
|- | |||
| BsmBI || 0.5 || 0.5 || 0.5 | |||
|- | |||
| dH<sub>2</sub>O || 5.25 || 5.25 || 5.25 | |||
|- | |||
| Total|| 10.0 || 10.0 | |||
|} | |} | ||
Latest revision as of 22:24, 26 September 2017
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1/29/13Type IIS Assembly Day 2
Purified PCR Product to 20fmol/μL
|