Cloning protocols
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The AGRIKOLA consortium has access to a set of 25000 Arabidopsis gene-specific tags (GSTs) designed for use in microarray studies. These GSTs, 150-600 bp in length, are ideal for targeted gene silencing in Arabidopsis if expressed from a suitable vector capable of producing double-stranded hairpin RNA (hpRNA). The basic two-step strategy for cloning GSTs into the hpRNA vector is depicted below.
Figure 1: The hairpin RNA strategy
- A. Amplification for adding the recombination site attB.
- B. Cloning of a GST as a Gateway™ entry clone by BP cloning.
- C. Cloning GSTs into the hpRNA vector.
- D. Predicted structure of the hairpin RNA when expressed in plant cells.
We will use high-throughput recombinational cloning to clone the GSTs into this vector thus generating up to 25000 plasmids, each of which can be used to reduce or eliminate expression of a single Arabidopsis gene by post-transcriptional silencing. These plasmids will be made available to the European (and worldwide) Arabidopsis community via the principal European Arabidopsis stock centre. We will also transform Arabidopsis with 4000 constitutive hpRNA clones and 1000 inducible hpRNA clones carrying chosen GSTs. Ten transformants carrying each construct will be analysed for obvious visible phenotypes before collecting seed. We will carry out more detailed analysis of a subset of these lines, concentrating on genes for which gene silencing is likely to be particularly appropriate. Seeds from the transgenic lines will be made available to the international Arabidopsis community via international stock centres and should prove an extremely valuable resource for functional analysis of Arabidopsis genes.
Protocols
PCR Amplification
| 10 x Red Cresol (Aldrich cat no. 11447-2) | 2 µl |
| 10 x PCR buffer | 2 µl |
| dNTP stock (25 mM of each dNTP) | 0.048 µl |
| column primer 10 µM | 1.6 µl |
| row primer 10 µM | 1.6 µl |
| Taq enzyme (home made...) | 0.4 µl |
| water | 11.952 µl |
| template (primary CATMA product) | 0.4 µl |
Templates correspond to primary GST amplicons. The quantity of template to use depends on the dilution of the amplicons. Quantities identical to the ones used for the secondary amplification of DNA spotted on microarrays are recommended.
PCR Conditions
| 94°C | 1 min | |
| 35 X | 94°C | 15 sec |
| 55°C | 15 sec | |
| 72°C | 30 sec | |
| 72°C | 5 min |
BP Cloning
| PCR product | 2 µl |
| pDONR 207 vector (50 ng/µl) | 1 µl |
| 5X BP clonase buffer | 1 µl |
| BP clonase | 0.4 µl |
| TE | qsp 5 µl |
| It's really important to add the mix to the PCR product and not the opposite ! | |
- Incubate reactions overnight at 25°C
- Add 0.5 µl of proteinase K (2 µg/µl)
- Incubate reactions 15 min at 37°C
Bacterial transformation in 96-well plates
- Add 10 µl of "ultra-competent" DH5α cells to 1 µl of BP reaction in 96-well PCR plates
- Incubate 20 min on ice-water
- Heat shock for 15 sec at 42°C in a water bath
- Incubate 5 min on ice-water
- Add 90 µl SOC media
- Shake 1 h at 37°C
- Add the 100 µl of transformed bacteria to 1 ml 2LB media 15 mg/l gentamycin in 2ml 96-well plates
- Shake at 37°C until OD=1.4; the bacterial density is critical for miniprep yields. We recommend 20 h at 800 rpm on a Heidolph Titramax 1000 shaker.
We recommend that you control transformation efficiency by plating say 4 samples on agar plates (for 4 wells add 2µl BP reaction + 20µl competent cells; add 180µl SOC and use 100µl for plating out the other 100µl for the liquid selection).
Inoue et al., 1990. Gene 96 : 23-28
PCR 2
using primers DNR5/DNR3
| DNR5 100 µM | 0.02 µl |
| DNR3 100 µM | 0.02 µl |
| 10 x Red Cresol (Aldrich cat no. 11447-2) | 1 µl |
| 10x PCR buffer | 1 µl |
| dNTP stock (25 mM of each dNTP) | 0.08 µl |
| Taq enzyme (home made...) | 0.2 µl |
| water | 7.18 µl |
| liquid selection bacteria | 0.5 µl |
PCR condition
| 94°C | 5 min | |
| 35 X | 94°C | 30 sec |
| 55°C | 30 sec | |
| 72°C | 2 min | |
| 72°C | 5 min |
Plasmid minipreps
Millipore Montage™ Plasmid Miniprep 96 Kit (Partial lysate protocol)
LR Cloning
| Miniprep plasmid DNA (75 ng/µl) | 1 µl |
| Destination vector | 75 ng |
| LR buffer 5X | 1 µl |
| LR clonase | 1 µl |
| TE | qsp 5 µl |
It's important to add the mix to miniprep plasmid and not the opposite !
- Incubate reactions overnight at 25°C
- Add 0.5 µl of proteinase K (2 µg/µl)
- Incubate reactions 15 min at 37°C
Bacterial transformation in 96-well plates
- Add 10 µl of "ultra-competent" DH5α cells to 1 µl of LR reaction in 96-well PCR plates
- Incubate 20 min on ice-water
- Heat shock for 15 sec at 42°C in a water bath
- Incubate 5 min on ice-water
- Add 90 µl SOC media
- Shake 1 h at 37°C
- Add the 100 µl of transformed bacteria to 1 ml 2LB media 50 mg/l kanamycin in 2ml 96-well plates
- Shake at 37°C until OD=1.4; the bacterial density is critical for miniprep yields. We recommend 20 h at 800 rpm on a Heidolph Titramax 1000 shaker.
We recommend that you control transformation efficiency by plating say 4 samples on agar plates (for 4 wells add 2µl BP reaction + 20µl competent cells; add 180µl SOC and use 100µl for plating out the other 100µl for the liquid selection).
Inoue et al., 1990. Gene 96 : 23-28
PCR 3
using primers Agri51/Agri56/Agri64/Agri69
| Agri51 100 µM | 0.02 µl |
| Agri56 100 µM | 0.02 µl |
| Agri64 100 µM | 0.02 µl |
| Agri69 100 µM | 0.02 µl |
| 10 x Red Cresol (Aldrich cat no. 11447-2) | 1 µl |
| 10x PCR buffer | 1 µl |
| dNTP stock (25 mM of each dNTP) | 0.08 µl |
| Taq enzyme (home made...) | 0.2 µl |
| water | 7.14 µl |
| liquid selection bacteria | 0.5 µl |
PCR conditions
| 94°C | 5 min | |
| 35 X | 94°C | 30 sec |
| 55°C | 30 sec | |
| 72°C | 2 min | |
| 72°C | 5 min |
Plasmid minipreps
Millipore Montage™ Plasmid Miniprep 96 Kit (Partial lysate protocol)
BglII digestions (optional)
- 500ng plasmid DNA
- 1 µl 10X NEBuffer 3
- 0.5 µl BglII (New England Biolabs, 10U/µl)
- water q.s.p. 10 µl
- Incubate 2hrs at 37°C