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. 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 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. | 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 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. | *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. | ||
Revision as of 10:19, 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 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.
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. | |
