Matt Gethers/CRI, Thailand/Labwork/Isolating HmgR/Week of 6.8.08

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6.11.08

Primer Design for HmgR ORF

Here is the KEGG File for HmgR (pa2010). I'd like to amplify this ORF and then drop it into the pET-11a commercial vector between the NdeI and BamHI sites. The ORF has to be in frame and I have to be sure there aren't any cloning sites in the ORF.

Using the file of HmgR from Mayuree

Forward Primer BT2736 (5' to 3')

Extra Bases (For cutting efficiency) NdeI Site Annealing Bases
gcg catatg gaaaagaacagcagtccc

Specs

LENGTH (annealing/all): 21/27

GC CONTENT (not including non-anealing bases):47.6 %

MELT TEMP (not including non-anealing bases): 55.4 ºC

Min(ΔG): -0.07 kcal.mole-1

Reverse Primer BT2737 (5' to 3')

Extra Bases (For cutting efficiency) BamHI Site Annealing Bases
cgc ggatcc gctcggagactgcgtcat

Specs

LENGTH (annealing/all): 18/27

GC CONTENT (not including non-anealing bases): 61.1 %

MELT TEMP (not including non-annealing bases): 57.4 ºC

Min(ΔG): -4.01 kcal.mole-1

References

pET-11a

pET-Blue2

pET System Manual