BBa_K187295

BBa_K187295 Version 1

Component

Source:
http://parts.igem.org/Part:BBa_K187295
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Stephen Jahns
Date created: 2009-10-19 11:00:00
Date modified: 2015-05-08 01:11:11

mrdA ORF, Forward Primer



Types
DnaRegion

Roles
primer

Primer

Sequences BBa_K187295_sequence (Version 1)

Description

This primer is for a gene deemed to be essential for a minimal E.coli genome by Team Biobytes. This primer is designed to amplify only the open reading frame, and produce ends that can be digested by restriction enzymes for insertion into an XbaI/PstI digested plasmid. These restriction sites were designed to be complementary to the restrictions sites found in pAB and pBA (PstI and XbaI). Due to the use of restriction enzymes, it was important to check each gene sequence for those same restriction sites. Genes which contained at least one PstI site, used the NsiI enzyme to be cloned into our standard plasmids (it produces the same overhang as PstI). Similarly if NsiI and PstI were in the gene sequence, SbfI was used. If all three of these sites were present in a gene sequence, other enzymes which produce the same overhang could be used. Genes which contained XbaI could use AvrII, NheI, as well as a multitude of others.

Notes

This primer produced a product of the predicted size using the following reaction conditions:

Water: 17.05uL

10x pfu buffer: 2.5uL

dNTPs (2mM): 2.5uL

DMSO: 1.2uL

MG1655 Genomic DNA: 0.5uL

Forward primer (10uM): 0.5uL

Reverse primer (10uM): 0.5uL

Pfu polymerase: 0.25uL

Total reaction volume: 25uL

Thermocycling conditions:

95C, 3min

95C, 30s

56C, 30s

72C, 3min

29 cycles to step 2

72C 2min
All Biobytes essential gene primers were produced using Vector NTI. Each primer's thermodynamic properties were examined using Vector NTI's hairpin and dimerzation programs. Primers were optimized to fit as many of the following criteria as possible:
Find the shortest possible sequence, reducing the cost to produce the primer.

Produce the highest score value possible.

Produce the closest Tm's possible

Produce hairpins with dG values >-5

Produce dimers with dG values >-10
The following are Vector NTI statistics for this primer:

dG Dimer (kcal/mol): -5.2

dG Hairpin (kcal/mol): -2.4

Source

Oligonucleotides were synthesized. Primers were tested using MG1655 E.coli genomic DNA as template.

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BBa_K187295/1