User:Mbennie/Notebook/Lab Notebook/Notebook/2007/08/16

Cellular Adhesion

 * PCR
 * Template: 40ul PCR Supermix, .4ul of each primer, and .4ul of PCR purification product
 * 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
 * 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)
 * Interesting: it appears that the PCR is the issue as the 200bp fragment was amplified solely from the correct length DNA fragment


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


 * Ligation
 * C: 6ul of C digest DNA, 7ul water, 1.5ul ligase buffer, .5ul ligase
 * D: 6ul of D digest DNA, 7ul water, 1.5ul ligase buffer, .5ul ligase
 * C + D: 6ul of C digest DNA, 6ul of D digest DNA, 1ul water, 1.5ul ligase buffer, .5ul ligase
 * B + C + D: 6ul of each digest DNA, 2ul ligase buffer, .5ul ligase
 * Protocol: 30mins@roomtemp,10mins@65C


 * Gel
 * Ran 1.5% gel for 30 minutes at 100V with samples
 * Ligations were completely used and mixed with about 5ul of loading dye
 * PCR products were used in normal proportions (5ul DNA, 2ul dye)
 * Looks like there is a ligation issue with part D


 * Oligos
 * 6His_Tag-F
 * 6His_Tag-R
 * FLAG_Tag-F
 * FLAG_Tag-R
 * Myc_Tag-F
 * Myc_Tag-R
 * HA_Tag-F
 * HA_Tag-R
 * GCN4-R2
 * Resuspended at 50uM


 * PCR
 * Template: 40ul PCR Supermix, .8ul primers (.4 primers for GCN4), .8ul DNA (only GCN4)
 * 6His Tag: 6His_Tag-F and 6His_Tag-R
 * FLAG Tag: FLAG_Tag-F and FLAG_Tag-R
 * Myc Tag: Myc_Tag-F and Myc_Tag-R
 * HA Tag: HA_Tag-F and HA_Tag-R
 * GCN4 Leucine Zipper: GCN4-F and GCN4-R2
 * Same protocol as 8.1.2007


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


 * Digest
 * Template: 3ul DNA (each), 2ul NEB2, .5ul Ear1, rest water (20ul rxn)
 * B+C
 * C+D
 * B+C+D
 * Thermocycler protocol: 1hr@37C, 20mins@80C


 * Gel
 * Ran 1.5% gel for 30 minutes at 100V with samples (5ul sample with 2ul of loading dye)
 * Everything looks good, will ligate into vector tomorrow


 * Ligation
 * Performed on digest tube above
 * Template (50ul rxns): 20ul digest, 5ul ligase buffer, .5ul ligase, rest water
 * Thermocycler protocol: 1hr@16C,10mins@65C


 * Gel
 * Ran 1.5% gel for 30 minutes at 100V with samples (15ul sample with 5ul of loading dye)
 * Looks promising, although very low ligation efficiency


 * PCR
 * Template: 40ul PCR Supermix, .4ul of each primer, and 1ul of ligation product
 * B+C: IgAb-F and Mut2_Pst1b-R
 * C+D: Mut_Pst1a-F and IgAb-R
 * B+C+D:IgAb-F and IgAb-R
 * B+C+D: BB_f and BB_r


 * Gel
 * Ran 1.5% gel for 30 minutes at 100V with samples (5ul sample with 2ul of loading dye)
 * A closer inspection reveals that cutting and ligating were successful (although the full construct was missing part C)
 * The rouge bands can be explained by incorrect hybridization of primers about 100bp from the end of part B
 * The two ligations involving two parts look good and are fairly clean -> see if adding last part works