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

DNA picked from gel (IgA primers), DNA picked from gel (BB primers), Complete IgAbc without muts, log-2 ladder

• 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

C (ligation), C (PCR), D (ligation), D (PCR), C + D (ligation), B + C + D (ligation), Complete IgAbc without muts, log-2 ladder

• 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

6His Tag, FLAG Tag, Myc Tag, HA Tag, GCN4 Leucine Zipper, log-2 ladder

• 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

B + C, C + D, B + C + D, log-2 ladder

• 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

B + C, C + D, B + C + D (IgA primers), B + C + D (BB primers), Complete IgAbc without muts, log-2 ladder

• 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