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NASC

The European Arabidopsis Stock Centre

SLAT insertion lines

Donated by

  • Ottoline Leyser Department of Biology, University of York
  • Jonathan Jones The Sainsbury Laboratory, John Innes Centre
  • Kanu Patel Department of Disease and Stress Biology, John Innes Centre
  • Sylvestre Marillonet The Sainsbury Laboratory, John Innes Centre
  • Ian Moore Department of Plant Sciences, Oxford University
  • Sean May Nottingham Arabidopsis Stock Centre (NASC), School of Biosciences, University of Nottingham
  • Caroline Dean Department of Cell and Developmental Biology, John Innes Centre

Click here to view all 4350 of these lines.

Description

Sets in this collection

Nasc code Description Set contents
N41980 SLAT set - seed - set 1 View set contents
N41981 SLAT set - seed - set 2 View set contents
N41982 SLAT set - seed - set 3 View set contents
N41983 SLAT set - seed - set 4 View set contents
N41984 SLAT set - seed - set 5 View set contents
N41985 SLAT set - seed - set 6 View set contents

Introduction

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 may be available from the Sainsbury laboratory Plant Molecular Genetics webpage.

DNA pools for the SLAT lines contain 50ng of DNA for the individual 50 line pools

This 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

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)

Primary PCR:

  • DNA 1µl
  • x10 PCR buffer 2µl
  • dNTP 2mM 2.5µl
  • Primer1 0.6µl
  • Primer2 0.6µl
  • Taq/Pwo* 0.2µl
  • dH2O 13.1µl
  • Total 20µl

* 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

  • 94°C 2min
  • 94°C 15sec
  • 60-65°C 30sec x35 cycles for the primary PCR
  • 68°C 4min (x25 cycles for the secondary PCR)
  • 68°C 5min

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 DNA isolated by your favourite method can be used.

Sequencing primers and direction:

Superpool Template generation Sequencing primer Recommended primer for PCR
01_x x_x x.txt
02_x x_x x.txt
03_x x_x x.txt
04_x x_x x.txt
05_x x_x x_1.txt

01_x x_x x b.txt
02_x x_x x b.txt
03_x x_x x b.txt
04_x x_x x b.txt
05_x x_x x_5.txt
Adaptor PCR - Mse I
Adaptor PCR - Mse I
Adaptor PCR - Mse I
Adaptor PCR - Mse I
IPCR - BstY I

Adaptor PCR - Bfa I
Adaptor PCR - Bfa I
Adaptor PCR - Bfa I
Adaptor PCR - Bfa I
IPCR - BstY I
spm3
spm3
spm3
spm3
dspm1

spm3
spm3
spm3
spm3
dspm5
dspm1
dspm1
dspm1
dspm1
dspm1

dspm1
dspm1
dspm1
dspm1
dspm5

**Note 1: All sequences for superpool 05 were generated from individual lines - all others were generated from pools.

**Note 2: In the SINS database, all dspm sequence has been removed from the flanking sequence.

Other recommended primers and positions in dSPM

Preferred primers - sequences

  • dspm1 CTT ATT TCA GTA AGA GTG TGG GGT TTT GG
  • dSpm8130: GAG CGT CGG TCC CCA CAC TTC TAT AC
  • dSpm8166: GTC CAT TTT AGA GTG ACG GCT AAG AGT G
  • dSpm8: GTT TTG GCC GAC ACT CCT TAC C
  • dspm5: CGG GAT CCG ACA CTC TTT AAT TAA CTG ACA CTC
  • dSpm11: GGT GCA GCA AAA CCC ACA CTT TTA CTT C

SLAT line numbering explained

Individual SLAT lines are numbered as XX_XX_XX for example 03_17_12. The last two digits specify the line number. The two digits to the left of this specify the pool number to which that line belongs, a total of 50 lines making up one pool. The first two digits specify the superpool to which that pool belongs, a total of 48 pools make up 1 superpool.

The majority of SLAT lines are distributed in their pools of 50. So for line 03_17_12 the orderable SLAT stock would be 03_17, which would contain a mix of all the seed lines within that pool.

Summary - 03_17_12

  • 03 = superpool number
  • 17 = pool number
  • 12 = line number

Important note regarding distribution of single lines in superpool 5

Limited supplies of seeds for single lines in superpool 05 are available thanks to Jonathan Jones, Sylvestre Marillonet and Kanu Patel.

These are for use in reverse genetics screens only. Forward screens would drain the resource at unacceptable levels of wastage for the limited numbers available (hit to miss ratio). We will be supplying approximately 200 seeds for each line and will unfortunately have to query large consecutively numbered orders.

Reverse genetics filters relating to this resource have previously been supplied by the Sainsbury Laboratory at the John Innes centre, Norwich, UK.

There is NO DNA currently available to release for these lines

We cannot distribute SETS of this material beyond any prior arrangements already being used to provide return bulkings of seeds and DNA to the stock centre for the reasons given above - please accept our sincere apologies.

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