User:Mbennie/Notebook/Lab Notebook/Notebook/2007/08/10

Cellular Adhesion

 * Liquid Culture
 * IgAbc #8 did not grow up correctly
 * New tube with 8ml Amp/Cl LB, inoculated from 8.9 colony PCR backup plate
 * Put in incubator at 8:30 AM


 * Glycerol
 * Took 1.6ml of liquid culture and created 10% glycerol stocks
 * GCN4 #1
 * GCN4 #3
 * Miniprep
 * Extracted DNA from remaining liquid culture (~6ml)
 * Eluted in 50ul water


 * Sequencing
 * 8.10.2007
 * All sequences were incorrect


 * PCR
 * Template: 40ul PCR Supermix, .4ul of each primer, and 1ul of ligation product
 * C + D with B with IgAb-F and IgAb-R
 * B + C with IgAb-F and Mut_Pst1b-R
 * C + D with Mut_Pst1a-F and IgAb-R
 * Same thermocycler protocol as 8.1.2007


 * 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
 * IgAss #3: 8ul miniprep DNA, .5ul EcoR1, .5ul Spe1
 * Fos #3: 8ul miniprep DNA, .5ul Xba1, .5ul Spe1
 * JunB #2: 6ul miniprep DNA, .5ul Xba1, .5ul Spe1
 * Protocol: 2hr@37C, 20mins@80C (only 1hr@37 necessary)


 * Gel
 * Ran 1.5% for 30 minutes at 100V to see if ligations worked
 * I think that Parts II and III are getting lost during PCR purification


 * Ligation
 * Template: .5ul 1AK3 vector, 1ul ligation buffer, .5ul ligase, 2ul of each digest product, rest water(10ul rxn)
 * IgAss #3 and Fos #3
 * IgAss #3 and JunB #2
 * 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 Kan(out of Amp/Kan plates) and grew up at 37C overnight


 * Antibody tags
 * Investigate creating antibody tags to see if IgA is being properly expressed on the surface of the cell
 * 6His (HHHHHH)
 * FLAG (DYKDDDDK)
 * Myc (EQKLISEEDL)
 * HA (YPYDVPDYA)
 * GST (220 aa)
 * All have Anti- from mouse