User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/27

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05/27/15

  • Rene - hi-res melt curve screen on CRISPR clones
  • Ben - continue luc replacement donor cloning



Rene - hi-res melt curve screen on CRISPR clones


  • Set-up
    • 2 replicates per clone
    • Total wells: 36 (1 well per rxn.)
    • Template+water Multiplier = none (use 10x diluted stock for all rxns)
    • Primer+SYBR Mix Multiplier = 27 + 3 = 39


  • Template+ Water: Dilute PCR reaction 10x
    • Use multichannel to transfer 5.0 μL PCR products into 45.0 μL dH2O
    • Set up dilutions in 8-tube strips
    • Use 4.5 μL Template+water per rxn
  1. Lu34_AA01 - 12
  2. Ga34_AA01 - 12
  3. nTc (3 wells)


  • Master Mix: Primers+SYBR
    • F = P149
    • R = P160
    • 10.5 per well
Reagent Volume (x30)
750 nM F/R primers 3.0 90.0
2x SYBR MM 7.5 225
  15.0


  • Plate loading
    • Pipette 21 Primers+SYBR (2 x 10.5) into first row (A1-12)
    • Multichannel-aliquot 10.5 Primers+SYBR into next row (B1-12)
    • Pipette 10.5 Primers+SYBR into C1-3 (nTc wells)
    • Multi-channel pipette 4.5 Template+water into appropriate wells (A1-12, B1-12)
    • Pipette 4.5 water into C1-3 (nTc wells)


  • Run PCR - Roche LC480: "SYBR Karmella CNV melt curve Template"
    • 95°C, 5 min
    • 30x [95°C, 10 sec; 60°C, 10 sec; 72°C, 10 sec (measure)]
    • Melt: continuous acquisition; 95°C, 4 sec; 70°C, 60 sec, -- +0.1°C/ 5 sec (programmed as "6 acquisitions/°C") --> 95°C
    • Cooling: 40°C, 30 sec


RESULTS:

  1. Lu34_AA01 - 12 -- Melt temps 85.1 ± 0.1 °C; additional short peak ~87°C
  2. Ga34_AA01 - 12 -- Melt temps 85.1 ± 0.1 °C; additional short peak ~87°C
  3. nTc (3 wells) -- no amplification
  • Try using more diluted template (100x)
  • Continue by sequencing a few purified PCR products with P215 to find a reference sample (wild type sequence)