User:Mbennie/Notebook/Lab Notebook/Notebook/2008/01/21: Difference between revisions
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** Same idea for GCN4 zipper | ** Same idea for GCN4 zipper | ||
** Use new primers (with extended cutting region) to start IGAbc pieces -> try to assemble B+C and C+D components | ** Use new primers (with extended cutting region) to start IGAbc pieces -> try to assemble B+C and C+D components | ||
** Clean out -80/-20/4 of old materials (unlabeled or useless) | |||
** Record 8/20/2007 sequence run results | |||
* Rest of the week | * Rest of the week |
Revision as of 15:04, 21 January 2008
Cellular Adhesion
- Review of all standard protocols used for this project
- PCR
- Template: 40ul PCR Supermix, .4ul of each primer, and 1ul of PCR product (or .4ul of N. gonorrhea DNA or .4ul PCR purification)
- Protocol:
- 5mins@95C
- 20secs@94C
- 20secs@55C
- 1min@68C
- REPEAT 2-4, 34 times
- 5mins@70C
- FOREVER@4C
- PCR Purification
- Used MinElute columns
- Eluted in 10ul of water
- Gel
- Ran 1.5% gel for 30 minutes at 100V with samples (5ul sample with 2ul of loading dye)
- Digest
- Template for IGA (single): 3ul DNA (each), 2ul NEB2, .5ul Sap1, rest water (20ul rxn)
- Template for IGA (pairs): 3ul DNA (each), 2ul NEB2, .5ul Ear1, rest water (20ul rxn)
- Template for tags: 3ul DNA (each), 2ul NEB2, .5ul EcoR1, .5ul Pst1, rest water (20ul rxn)
- Thermocycler protocol: 1hr@37C, 20mins@80C
- Construct Digest
- Beginning to assemble parts into a functional construct
- Template: 200ng DNA, 2ul NEB2, .2ul BSA, .5ul of each enzyme, rest water (20ul)
- The first part should be cut with EcoR1 and Spe1
- The second part should be cut with Xba1 and Pst1
- Ligation
- 6ul of IGA digest DNA, 7ul water, 1.5ul ligase buffer, .5ul ligase
- Template for IGA (assemble): .2ul 1AC3 vector, 1ul ligation buffer, .2ul ligase, 1ul digest product, rest water(10ul rxn)
- Protocol: 30mins@roomtemp,10mins@65C
- Transformation
- 50ul of TOP10 cells on ice with 2ul of DNA for 30mins
- 45secs heat shock at 42C using water bath
- Added 300ul of SOC
- Incubated for 1hr at 37C
- Plated on Amp/Cl and grew up at 37C overnight
- Liquid Culture
- Inoculated 8ml Amp/Cl LB with antibody tags and GCN4 from plate colonies
- Grew up in 37C overnight
- Glycerol
- Took 1.6ml of liquid culture and created 10% glycerol stocks
- Miniprep
- Extracted DNA from remaining liquid culture (~6ml)
- Eluted in 50ul water
- Sequencing
- Template: 3ul Miniprep DNA, .3ul primer, 6.7ul water
- Template: 1ul PCR DNA, .3ul primer, 8.7ul water
- 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
- PCR
- Status
- IGA
- beta-core: incomplete
- signal sequence: complete (made on 8/5/2007, in freezer box position 1.3)
- Leucine Zippers
- GCN4 Leucine Zipper: incomplete (due to bad primers)
- Fos Zipper: complete (made on 8/6/2007, in freezer box position 1.4)
- JunB Zipper: complete (made on 8/6/2007, in freezer box position 1.5)
- Tags
- 6His Tag: incomplete
- FLAG Tag: incomplete
- Myc Tag: incomplete
- HA Tag: incomplete
- IGA
- Plan for tomorrow
- Start from scratch with tag primers (3 of each) -> get all the way to cultures
- Same idea for GCN4 zipper
- Use new primers (with extended cutting region) to start IGAbc pieces -> try to assemble B+C and C+D components
- Clean out -80/-20/4 of old materials (unlabeled or useless)
- Record 8/20/2007 sequence run results
- Rest of the week
- Sequencing submitted goals:
- Tags (Wednesday)
- Zipper (Wednesday)
- IGAbc (Friday)
- Sequencing submitted goals: