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Protocol for the validation of the GST entry clones and the deposit of these clones in the BCCM/LMBP Plasmid collection

Click on 'figure numbers' or on the words 'details' to read the related description.

GST clones validation schema Start Figure 1 Figure 2 Figure 3b Figure 3a Figure 4 Figure 5 Figure 6 Figure 8 Figure 7 Figure 11 Figure 9 Figure 10 Start Figure 1 Figure 2 Figure 3b Figure 3a Figure 5 Figure 4 Figure 6 Figure 8 Figure 7 Figure 11 Figure 10 Figure 9

Description

START Bacterial clones
  The starting material is a collection of bacterial mixes obtained from the partners of the AGRIKOLA project (Belgium, UK, France and Spain) and delivered in multi-well plates.
Figure 1 Plating and growth of bacterial mix
  To obtain well isolated colonies for each entry clone, starting from the original bacterial mixes, one drop of each clone mix is inoculated and spread on a agar plate with solid LB+ medium with gentamicin.

After incubation, the purity of the culture is checked.
Figure 2 Picking and subcultivation of 4 individual bacterial colonies
  If the purity check is positive, for each entry clone 4 independent colonies are picked and transferred in standard 96-well plates.

The viability is checked after incubation.
Figure 3a/b Cell lysis and glycerol stock

 

 A fraction of the 4 subcultures is used to make a lysate and simultaneously 75μ of the subcultures is transferred in 96-well plates filled with 75 μl sterile glycerol to obtain a glycerol stock which will be temporarily stored at –80°C.
Figure 4 PCR-testing
 
  • As a first step in confirming the identity of the GST of interest, the insert in the recombinant plasmids carried by the four selected colonies is tested by PCR.

  • The primers used for this step are derived from the pDONR207 (5,584 bp) vector backbone and are located close to the attL sites.

  • The PCR results are analyzed by DNA agarose gel electrophoresis and reveal the bacteria containing empty vectors or a wrong or unexpected GST showing an unexpected size.

  • A size <700 bp indicates the absence of a GST insert; no sequence analysis will be done on such 'empty' vectors. A size within the range 'theoretical GST ±20%' is considered good.

  • Only one positive clone out of the four originally picked is selected for further validation.
Figure 5 Subcultivation of the selected clone from the glycerol stock

 
  • The entry clone selected in the previous step is subcultivated on solid LB+ medium with gentamicin, starting from the glycerol stock described in Figure 3b.

  • After incubation, the purity of the culture is checked. The purity check is one of the 3 quality control tests of the BCCM/LMBP quality management system for the deposit of plasmids in the public collection.
Figure 6 Subcultivation of a single colony from the LB+ agar plate

 
  • One single colony is subcultivated in Greiner tubes, starting from the LB+ agar plate described in Figure 5.

  • After incubation, the viability of the culture is checked. The viability check is one of the 3 quality control tests of the BCCM/LMBP quality management system for the deposit of plasmids in the public collection.
Figure 7 reparation and storage of the host/plasmid combination
  If the viability check is positive, 3 x 0.5 ml of the subculture is transferred in 3 NUNC tubes with 0.5 ml glycerol:
  • 2 NUNC tubes are stored in the BCCM/LMBP stock: 1 in the master stock and 1 in the distribution stock;

  • 1 NUNC tube is stored in the PSB stock.
Figure 8 Preparation of DNA for authenticity test and cryopreservation
  If the viability check is positive, plasmid DNA is prepared using a robotic platform programmed for this purpose. The protocol is a variation of the alkaline lysis procedure in which the successive steps are performed with an 8-channel liquid handling tool, on a Biomek 2000 Laboratory Automation Workstation, using the Wizard Magnesil Plasmid DNA purification system (Promega).
Figure 9

PCR-testing

 
  • As an extra verification, the plasmid DNA is checked for contamination by a PCR reaction. Simultaneously, the PCR analysis is used to confirm insertion of the attL1-GST-attL2 cassette into the entry clone.

  • A size <700 bp indicates the absence of a GST insert; no sequence analysis will be performed on such plasmids. A size within the range theoretical GST ±20% is considered good.
Figure 10 Authenticity check by DNA-sequencing + BLAST

 
  • The clean PCR products are sequenced with one BigDye (ABI) reaction and the resulting profile is analyzed with an ABI 3700 capillary automated sequencer.

  • A single sequence reaction and run will be sufficient per clone since the GSTs are 150 to 500 base pairs in length and sequencing runs are routinely informative over 800 bp.

  • Authenticity is checked by blastn homology searches between the experimental DNA sequence derived from the PCR samples and all GSTs sequences in the CATMA database.An alignment length >=60 bp and a DNA sequence homology >95% is considered as a positive confirmation of the GST identity.

  • Small insertion/deletion or point mutations potentially introduced by the successive PCRs are not problematic. The molecules responsible for RNAi are 21 to 23 nt-long small interfering RNAs that are chopped from the larger GST-derived hairpin RNA. Therefore a few point mutations in a GST will not alter its ability to induce silencing as they will not affect the sequence of most siRNAs.

The authenticity check, together with the PCR test, is the third quality control test of the BCCM/LMBP quality management system for the deposit of plasmids in the public collection.
Figure 11 Preparation and storage of DNA

 
  • When the authenticity of the entry plasmid clones can be established, the remaining DNA, prepared in Figure 8, is transferred in 2 multi-well plates and precipitated with ethanol.

  • The two multi-well plates are stored at −20°C on separate locations.
  Data input
 
  • To create and complete the database record, a scientific collaborator of BCCM/LMBP checks for each validated GST entry clone whether all necessary data have been provided by PSB to BCCM/LMBP.

  • Creation, completion and finalization of the database record, datasheet and circular map is done by a scientific collaborator of BCCM/LMBP.