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

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Cellular Adhesion

DNA picked from gel (IgA primers), DNA picked from gel (BB primers), Complete IgAbc without muts, log-2 ladder
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
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
  • 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
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
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
B + C, C + D, B + C + D (IgA primers), B + C + D (BB primers), Complete IgAbc without muts, log-2 ladder
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
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