Prbbbb:vector pcr

Overview

PCR reaction to generate linearized construction vector backbones for Fusion(Freiburg)-formatted Biobrick assemblies.

Materials

• 100 ul + PCR tubes
• Phusion HotStart Polymerase 2 U/ul
• Phusion HF Buffer 5x
• dNTP mix 10mM each nucleotide
• ddH2O
• reverse prefix primer rg0301 (fusion format)
• reverse suffix primer rg0302 (fusion format)
• vector template DNA
  • pSB1AC3F -- BBa_J18901
  • pSB1AK3F -- BBa_J18902
  • pSB1AT3F -- BBa_J18903

Procedure

1. Step 1: setup PCR reaction

	µl, single reaction	3.5xMaster	10xMaster
H2O	76µl	266	760
5x HF Buffer	20µl	70	200
10mM dNTP	2µl	70	200
rg0301 100 µM	0.5µl	1.75	5
rg0302 100 µM	0.5µl	1.75	5
Phusion	1µl	3.5	10
template DNA	0.1µl	0.35	1

1. Step 2
   • Step 2 has some additional information that goes with it. i.e. Keep at 4°C.
2. Step 3
   1. Step 3 has multiple sub-steps within it.
   2. Enumerate each of those.

Notes

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References

Relevant papers and books

1. Goldbeter A and Koshland DE Jr. An amplified sensitivity arising from covalent modification in biological systems. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6840-4. DOI:10.1073/pnas.78.11.6840 |
2. JACOB F and MONOD J. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol. 1961 Jun;3:318-56. DOI:10.1016/s0022-2836(61)80072-7 |
3. ISBN:0879697164

Contact

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