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

(Difference between revisions)

Revision as of 03:02, 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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@@ Line 14: / Line 14: @@
 ** 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.