User:Mbennie/Notebook/Lab Notebook/Notebook/2007/08/19: Difference between revisions
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*** 68C for 10 mins | *** 68C for 10 mins | ||
*** 4C FOREVER | *** 4C FOREVER | ||
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* Gel | * Gel | ||
** Ran 1.5% for 30 minutes at 100V to see if PCR product is correct length (5ul of PCR product with 2ul of dye) | ** Ran 1.5% for 30 minutes at 100V to see if PCR product is correct length (5ul of PCR product with 2ul of dye) | ||
Revision as of 15:49, 19 August 2007
Cellular Adhesion
- Liquid Culture
- Inoculated 8ml Amp/Cl LB with antibody tags and GCN4 from plate colonies
- Inoculated 8ml Amp/Kan LB with autotransporter from plate colonies
- Put in incubator at 12:15 AM
- Colony PCR
- Template: 20ul Supermix, .8ul VF2 and VR, 1 ul of cell dilution (colony in 100ul of water)
- Picked three colonies from each plated sample to PCR
- B+C w/ D (IgA primers)
- C+D w/ B (IgA primers)
- B+C w/ D (BB primers)
- C+D w/ B (BB primers)
- 6His Tag
- FLAG Tag
- Myc Tag
- HA Tag
- GCN4 Leucine Zipper
- Grew up liquid cultures of all samples for miniprepping (if gel shows correct insert)
- Protocol:
- 95C for 15 mins
- 94C for 30 secs
- 55C for 30 secs
- 68C for 1.5 min
- REPEAT 2-4 39 times
- 68C for 10 mins
- 4C FOREVER
- Gel
- Ran 1.5% for 30 minutes at 100V to see if PCR product is correct length (5ul of PCR product with 2ul of dye)
