Strains, Plasmids and Primers
We are happy to send you strains to get recombineering working in your lab. We also are happy to send you plasmids as transformants. You can mini-prep plasmid DNA and then transform them into your bacterial strain of choice to enable recombineering. To date, we have sent out more than 5000 orders for strains and plasmids! Ask for them by strain or plasmid number, but please ask only for what you really need. Maintaining our strain collection is time consuming and expensive for us. If you want strains specifically for doing BAC work, see this site first. Strains and plasmids can be requested by sending us an email. With your request, please include your FEDEX number, your phone number and your address to expedite shipping. If you are a company and would like some of our strains, we must have you contact Rose Freel (rose.freel@nih.gov) first at the NIH Office of Technology Transfer. Thanks!
For your convenience, the below strain list with complete genotypes can be downloaded as a Word file by clicking Recombineering Strains.docx.
The list of plasmids is below the strain list. Plasmids are in DH10B.
STRAINS
| STRAIN | GENOTYPE | SPECIAL PURPOSE |
|---|---|---|
| LT521 | MG1655 gal490 nadA::Tn10 pglλ8 [λ cI857 λ(cro-bioA)] | Useful for moving prophage into other genetic backgrounds by P1 transduction using linked Tn10. |
| DY329 | W3110 λlacU169 gal490 nadA::Tn10 pglλ8 [λ cI857 λ(cro-bioA)] | Useful for moving prophage into other genetic backgrounds by P1 transduction using linked Tn10. |
| DY330 | W3110 λlacU169 gal490 pglλ8 [λ cI857 λ(cro-bioA)] | |
| DY331 | W3110 λlacU169 gal490 pglλ8 [λ cI857 λ(cro-bioA)] λ(srlA-recA)301::Tn10 | Recombineering targeting a plasmid |
| DY378 | W3110 [λ cI857 λ(cro-bioA)] | |
| HME6 | W3110 galKtyr145UAG λlacU169 [λ cI857 λ(cro-bioA)] | This strain contains the galK assay system for oligo recombination. It is a useful control to verify that your oligo recombination is working. |
| SIMD50 | HME6 [λ cI857 (int-cIII<>bet)] | "Beta only" strain. Great for oligo recombination. Will not work for dsDNA recombination as it has no exo or gam. |
| SIMD90 | SIMD50 mutS<>cat | High frequency oligo recombination. Defective for MMR (methyl-directed mismatch repair). |
| SIMD62 | HME6 [λ cI857 (int-cIII<>cat exo bet) λ(cro-bioA)] | This strain has no gam gene and thus avoids toxicity associated with it. However, recombination with dsDNA is reduced ~10-fold. The beta gene is fused to the start codon of cIII. |
| HME63 | HME6 mutS<>amp | Defective for MMR thus gives high frequency oligo recombination. Oligo recombination to Gal+ can be used as a control. |
| HME68 | HME6 mutS<>cat | Defective for MMR thus gives high frequency oligo recombination. Oligo recombination to Gal+ can be used as a control. |
| HME70 | HME6 mutS<>cat λ(srlA-recA)301::Tn10 | High frequency oligo recombination when targeting plasmids. Defective for MMR. Also works for dsDNA recombination. |
| HME71 | HME6 λ(srlA-recA )301::Tn10 | Oligo or dsDNA recombination with plasmids. |
| T-SACK | W3110 araD<>tetA-sacB-amp fliC<>cat argG::Tn5 | Template for tetA, kan, amp, cat, and tetA-sacB cassettes, amplify with colony PCR. |
| PLASMID | ORIGIN | DRUG RESISTANCE |
|---|---|---|
| pSIM5 | pSC101 repAts | Chloramphenicol |
| pSIM6 | pSC101 repAts | Ampicillin |
| pSIM7 | pBBR1 | Chloramphenicol |
| pSIM8 | pBBR1 | Ampicillin |
| pSIM9 | pRK2 trfAts | Chloramphenicol |
| pSIM10 | pRK2 trfAts | Hygromycin |
| pSIM17** | pSC101 repAts | Blasticidin |
| pSIM18** | pSC101 repAts | Hygromycin |
| pSIM19 | pSC101 repAts | Spectinomycin |
| pSIM27 | pSC101 repAts | Tetracycline |
| pSIM29 | pSC101 repAts | Hygromycin |
PLASMIDS
** Be aware that these plasmids have homology to the AmpR cassette, sequence flanking the drug resistance gene but are not AmpR. This can cause unexpected recombinants if you are targeting an amp-cassette into cells containing this plasmid. Use a different pSIM plasmid, such as pSIM29, if you are targeting with an AmpR cassette.
PRIMER SEQUENCES
cassette primers 2016
SEQUENCE FILES
Below are sequence files of the plasmids and other useful sequences in three formats, “pdf”, “text” or GCK. The GCK files require Gene Construction Kit from Textco to view. You can download a demo here. Also, a free version of SnapGene, SnapGene Viewer, will read these files. It can be downloaded here.