The Slat collection
The SLAT lines (1) were generated by Dr Jonathan Jones, Sainsbury Laboratory, John Innes Centre, Norwich, UK.
The background of this population is Columbia (Col-0 : Stock number N1092). The binary vector used to generate the lines contains a T-DNA that carries (i) a non-autonomous Spm derivative containing the BAR gene selectable marker, (ii) a counter selectable genetic marker based on a bacterial cytochrome P450 (2) that activates a DuPont proherbicide and (iii) an Spm transposase (TPase) gene under the control of the P35S, pSpm or meiosis-specific pAtDMC1 (3) promoters. This construct permits the selection for stable, unlinked with T-DNA, single copy insertions.
Further information about the Slat lines and links to the SINS database are available from the Sainsbury laboratory Plant Molecular Genetics webpage
DNA pools for the SLAT lines contain 50ng of DNA for the individual 50 line poolsThis should be resuspended in 5µl water or T.E. before following the protocol given below.
(this is enough for one screen with a control - you are only expected to use this DNA to confirm that the seed pool is correct for your purposes).
PCR screening of the Slat DNA
Requirements for primers:
For each gene of interest 4 gene-specific primers are required with Tm above 60°C : two primers flanking the known sequence and two nested. Hybridisation to a blot of the primary PCR will give an indication of absence or presence of the gene of interest, if the later is the case a fully nested PCR can be carried out for confirmation.
dSpm 3' end
dspm1: CTT ATT TCA GTA AGA GTG TGG GGT TTT GG (Tm:67.9°C)
nested dspm8: GTT TTG GCC GAC ACT CCT TAC C(Tm: 68°C)
dSpm 5' end
dspm11: GGT GCA GCA AAA CCC ACA CTT TTA CTT C(Tm: 69°C)
nested dspm5: CGG GAT CCG ACA CTC TTT AAT TAA CTG ACA CTC (Tm: 68.0°C)
* A mixture Taq:Pwo in a unit ratio of 160:1 is used in order to amplify fragments of up to 5 or 6kb.
Secondary nested PCR
Dilute the product from the 1st PCR 1:10, and use 1μl for a 20μl reaction with the appropriate primer combination.
PCR Cycling parameters
The PCR reactions should be run with the dSpm-specific primers in all possible combinations using as the templates DNA from all superpools available. The PCR products can then be checked by Southern analysis using a gene-specific probe. In case of positive superpool identification, PCR screen should be carried out on DNA pools of positive superpool with the appropriate pair of dSpm- and gene-specific primers in order to identify the positive pool of 50 lines.
Approximately 500 seeds from the positive pool should be germinated in the glasshouse, sprayed with a mix of 100mg/L phosphinotricin, plus 50ml/L of Silwet in order to remove all plants without the dSpm insertion. Surviving plants can then be used for PCR screen of mutant. As a template either DNA isolated by your favourite method or plant tissue (4) can be used.
1. Tissier, A. et al. Multiple Independent Defective Suppressor-mutator Transposon Insertions in Arabidopsis: A Tool for Functional Genomics, Plant Cell. (1999) 11(10):1841-52.
2. O'Keefe, D.P. et al., Plant Physiol. (1994) 105:473-482.
3. Klimyuk, V.I. & Jones, J.D.G. Plant J. (1997) 11:1014.
4. Klimyuk, V.I. et al., Plant J. (1993) 3:493-494).