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Strategies and Results of Routine Semi-automated Cloning

Strategies and Results of Routine Semi-automated Cloning. Heath Klock - JCSG. Our Vector Strategy. Small, non-cleavable expression/purification tag directly followed by the gene of interest Blunt/Pac strategy directs orientation while eliminating extra codons/residues in the N-terminal tag

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Strategies and Results of Routine Semi-automated Cloning

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  1. Strategies and Results of Routine Semi-automated Cloning Heath Klock - JCSG

  2. Our Vector Strategy • Small, non-cleavable expression/purification tag directly followed by the gene of interest • Blunt/Pac strategy directs orientation while eliminating extra codons/residues in the N-terminal tag • Toxic ccdB protein product is expressed in transformants that contain undigested or religated vector background Para PT7 Sma I Pme I 6-aa 6-his RBS CAC GTG Cm/ccdB TTA ATT AA Nco I Pml I Pac I

  3. Our Oligo Strategy • 5’ Primer • Begins with the first codon of the reading frame to be expressed • 3’ Primer • Contains a stop codon, an insert-specific 6 base “SYBR” sequence, and a Pac I restriction site • Strategy allows for the same PCR product to be ligated into various Blunt/Pac-adapted vectors Gene Product of Interest Stop SYBR Pac I

  4. MWG Biotech - Roboseq 4204 SE Liquid and plate handling for PCRs, clean-ups, digests and ligations Bio-tek - Synergy HT Microplate reader for absorbance and fluorescence, kinetic and endpoint assays Our robotics…

  5. Primus HT – Multi-block thermocyclers Motorized lids PCRs, digests and ligations ST Robotics – R16 robot arm Transfers plates between main deck, plate reader, thermocyclers and off-deck storage Two more pieces…

  6. The robotics have been integrated to create “Tabitha” 6 ft. Synergy HT Plate Reader Primus HT Thermo cyclers 6 ft. R16 arm RoboSeq Liquid Handling Deck Off deck Plate Storage

  7. Day 1 Initial PCR setup Gel Analysis* PCR Cleanup  Pac I digest Ligations Transformations  Day 2 Colony Picking  Day 3 Diagnostic PCR setup* SYBR assay* Reracking positives* Exo/SAP Sequence Later Analyze sequence* Final glycerol stocking Process and Timeline for 96 Targets  not currently automated Stagger start “Day 1” up to 4 times per week for attempting ~384 targets in a week

  8. Workload requirements per plate of targets PCR, cleanup, Pac digest (Day One) Ligation, transformation (Day One) Colony picking (Day Two), dPCR, SYBR (Day Three) Rerack, Exo/SAP, sequence (Day Three) Final glycerol stock archive (Later)

  9. Gel Loading and Documentation by Tabitha and E-gels • Samples of completed PCRs are loaded onto an E-gel 96 (Invitrogen) along with a DNA ladder • E-gel 96 Editor (Invitrogen) converts the raw image into a well-labeled, notebook-ready image

  10. Diagnostic PCR • Cells from cultures of isolated colonies are used directly as template for automated dPCR reaction setup. No minipreps!! • A 5’ vector-specific primer is used with a 3’ primer that is universally specific to insert-containing plasmids only • PCR works only when an insert is present Insert present. PCR product accumulates!! GOI No insert. No PCR product. No GOI

  11. SYBR Assays and Amplicon Reracking • Fluorescence intensity of SYBR Gold is enhanced when bound to double-stranded PCR product vs. single-stranded oligonucleotides • Wells with accumulated PCR product (vector with insert) fluoresce ~10-fold more intensely than those wells without PCR product (vector without insert) • An Excel spreadsheet identifies the two most fluorescent SYBR-positive wells for each putative clone and creates rerack files for consolidating amplicons for Exo/SAP reactions and submission to sequencing • 80-90% correlation to running gels. 10 times faster!!!

  12. Sequence Analysis • Reracked PCR products are submitted to sequencing. Again, no minipreps!! • ABI sequence trace files are converted to FastA files by Seqman (DNAStar) • FastA sequence files are uploaded into CloneCompare (GNF) • CloneCompare finds the 5’ 6thio/6his tag, BLASTs the adjacent, downstream sequence against a pre-compiled database of clone candidates and returns the clone’s identity as well as n-1 truncation information 6thio/6his Gene of Interest CloneCompare

  13. Cloning Results to Date

  14. Conclusions • Developed a highly, not fully, automated platform for cloning • Requires no minipreps and only one DNA gel of 96 samples for every 480 PCR reactions • The use of SYBR assays and CloneCompare facilitates fast, “hands-free” identification of expression-ready clones • Large numbers of clones can be generated quickly with success rates similar to manual cloning using only one person’s time

  15. Aprilfawn White Tabitha specialist Tanya Shin Cloning Scott Lesley Program Head Tony Orth Cloning Strategies Keith Ching Sequence Analysis Michael Winniman MWG robotics integration NIH PSI P50 GM62411 Acknowledgements

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