User:Mbennie/Notebook/Lab Notebook/Notebook/2008/01/21

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


 * 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


 * 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)