User:Mbennie/Notebook/Lab Notebook/Notebook/2007/08/15

Cellular Adhesion

 * PCR Purification
 * Used MinElute columns to PCR purify tube D
 * Eluted in 10ul of water


 * Digest
 * B + C with D: 3ul D DNA, 3ul B + C DNA, 2ul NEB4, .5ul Sap1, rest water (20ul rxn)
 * Thermocycler protocol: 1hr@37C, 20mins@80C


 * Ligation
 * Performed on digest tube above
 * Template (50ul rxns): 20ul digest, 5ul ligase buffer, .5ul ligase, rest water
 * Thermocycler protocol: 30mins@roomtemp,10mins@65C


 * PCR
 * Template: 40ul PCR Supermix, .4ul of each primer, and 1ul of ligation product
 * B + C with D: IgAb-F and IgAb-R
 * B + C with D: BB_f and BB_r (.8ul of each)
 * Same thermocycler protocol as 8.1.2007


 * Gel
 * Ran 1.5% gel for 30 minutes at 100V with samples (5ul sample with 2ul of loading dye)
 * Didn't work, but new primers are here anyway


 * Oligos
 * Mut2_Pst1b-F
 * Mut2_Pst1b-R
 * Resuspended at 50uM


 * PCR
 * Template: 40ul PCR Supermix, .4ul of each primer, and .4ul N. gonorrhea DNA
 * B: Part 1 of IgA beta-core IgAb-F and Mut_Pst1a-R
 * C: Part 2 of IgA beta-core Mut_Pst1a-F and Mut2_Pst1b-R
 * D: Part 3 of IgA beta-core Mut2_Pst1b-F and IgAb-R
 * E: Full IgA beta-core IgAb-F and IgAb-R
 * Same protocol as 8.1.2007


 * Gel
 * Ran 1.5% gel for 30 minutes at 100V with samples (5ul sample with 2ul of loading dye)
 * Everything looks good, proceeding to put the pieces together


 * PCR Purification
 * Used MinElute columns to PCR purify tubes B, C, and D
 * Eluted in 10ul of water


 * Digest
 * B + C + D: 3ul DNA (each), 2ul NEB4, .5ul Sap1, rest water (20ul rxn)
 * Thermocycler protocol: 1hr@37C, 20mins@80C


 * Spec
 * B: 67.5 ng/ul
 * C: 54 ng/ul
 * D: 100.2 ng/ul


 * Ligation
 * Performed on digest tube above
 * Template (50ul rxns): 20ul digest, 5ul ligase buffer, .5ul ligase, rest water
 * Thermocycler protocol: 30mins@roomtemp,10mins@65C


 * PCR
 * Template: 40ul PCR Supermix, .4ul of each primer, and 1ul of ligation product
 * B + C + D: IgAb-F and IgAb-R
 * B + C + D: BB_f and BB_r (.8ul of each)
 * Same thermocycler protocol as 8.1.2007


 * Gel
 * Ran 1.5% gel for 30 minutes at 100V with samples (5ul sample with 2ul of loading dye)
 * It didn't work, what a surprise


 * PCR Purification
 * Used MinElute columns to PCR purify tubes B, C, and D
 * Eluted in 10ul of water
 * Messed up and eluted in catch tube, definitely contaminated DNA with some PE buffer
 * Used for digest, threw away the rest of the DNA


 * Digest
 * Template (20ul rxn): 3ul B + C + D DNA, 2ul NEB2, .2ul BSA, .5ul EcoR1, .5ul Pst1, rest water
 * Done for both the PCRs (one using IgA primers, the other using BB primers)
 * Protocol: 1hr@37C, 20mins@80C
 * PCR didn't amplify correct length band, threw digest away


 * PCR
 * Poked the gel where the correct length DNA segment should be and dipped in Supermix for template
 * Template: 40ul PCR Supermix, and .4ul of each primer
 * B + C + D: IgAb-F and IgAb-R
 * B + C + D: BB_f and BB_r (.8ul of each)