Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Zach Wiltshire
Date created: 2009-10-19 11:00:00
Date modified: 2015-05-08 01:11:11
rplD ORF, Reverse Primer
| Types | DnaRegion |
| Roles | Primer primer |
| Sequences | BBa_K187282_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:
95oC, 3min
95oC, 30s
56oC, 30s
72oC, 3min
29 cycles to step 2
72oC 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): (from the spreadsheet)
dG Hairpin (kcal/mol): (from the spreadsheet)