Haynes Lab:Notebook/Engineering PC-TFs/2013/01/29

(Difference between revisions)

Revision as of 03:01, 1 February 2013

Engineering PC-TFs

Digestion/ ligation reaction

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

Perform BsmBI/ T4 ligase mediated assembly

Reagent	Vol.	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
dH₂O	0.25
	10.0 μL

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 <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
 ==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
+|-
+| &nbsp; || 10.0 μL
+|}
+|}