Shreffler:Notebook/Alex Daily/2012/03/30: Difference between revisions
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==EoE Microbiome / TCR sequencing troubleshooting== | ==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 http://tools.invitrogen.com/content/sfs/manuals/topota_man.pdf) are not found next to each other | 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 http://tools.invitrogen.com/content/sfs/manuals/topota_man.pdf) are not found next to each other. Since the supposedly invariable vector sequences are 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 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. | |||
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Revision as of 10:12, 30 March 2012
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EoE Microbiome / TCR sequencing troubleshootingTCR 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 http://tools.invitrogen.com/content/sfs/manuals/topota_man.pdf) are not found next to each other. Since the supposedly invariable vector sequences are 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 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.
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