Shreffler:Notebook/Alex Daily/2012/03/30

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EoE Microbiome / TCR sequencing troubleshooting

TCR sequencing from this week yielded questionable sequences from Genewiz. For one thing, the BstX I and EcoR I sequences on the pCR 2.1-TOPO Map (page 21 here are not found next to each other. Since the vector sequence is not present in order, Moshe believes that the TOPO vector (2.1) is old and degraded - vector is linear and subject to end degradation, and if new ends are complementary to each other, can close the loop. Even though we don't supply the system with a ligase to seal loop, this can be from host bacterial origin. Thus, despite no insertion and no supposed plasmid, a plasmid is formed and allows for viable colonies.

  • Invitrogen technical support suggests that E. coli (and other bacteria) commonly rearrange plasmids, causing partial or complete excision/rearrangement of inserted PCR product. To minimize chance of this occurring, transfected cells AND picked colonies can be cultured at lower temperatures (RT), thus slowing cell processes including plasmid rearrangement.
  • Further suggestions include choosing different cloning strains to transfect, including DH5-alpha and TOP10. Another suggestion is "stable cell" line, with product number 10268019 (need to research on Invitrogen website).
  • If all else fails, use both stable cell line and culturing at RT.

Moshe's spontaneous plasmid formation due to host enzyme interference is corroborated by Invitrogen tech support's information. However, he doesn't believe that plasmid excision/rearrangement has occurred, due to the small size (~600 bp) of our insertion (rearrangements are much more likely with larger (5000 bp) insertions. Furthermore, shelf life of TOPO 2.1 Vector is 6 months after receipt, and we are nearing 5 months, with constant thawing and freezing. Instead of using new TOPO and/or culturing at RT, however, Moshe would like to try transfection/cloning using the PGem kit, which we plan on doing today.

Another possibility that Moshe mentioned for why cloning might have failed is that the amplified insertion region must have T/A overhangs in order to be inserted, and if any end degradation occurs, insertion may not happen successfully.

pGEM-T Easy Vector System I

Started pGem system using protocol adapted from Douek et al ("Unbiased Molecular Analysis of T Cell Receptor Expression Using Template-Switch Anchored RT-PCR"). Protocol calls for gel-extracted PCR product.

Amplicon ligation calls for overnight incubation (12-18 hr) at 4°C, so I'll remove and freeze them tomorrow morning.

Splitting cells for Bert

Only orange wells were "Unstimmed" and "alpha-CL? Protein," (two wells each). Resuspended cells by pipetting up and down. Split by pipetting half volume (1 ml) into new well in same row, and adding 1 ml fresh medium (AIM V + 20U/ml IL-2) in all wells. (Note: medium wasn't warmed to 37°C; came straight from 4°C).