User:Karmella Haynes/Notebook/BioBrick cloning/2015/06/22

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06/22/15

  • Rene - CRISPR PCR library PCR
  • Cas-tone - PCR new inserts



Rene - CRISPR PCR library PCR

  • Part 1 - GoTaq "Dirty" PCR on liquid mini-cultures, 600 bp amplicons; primers P163/P215
  • Part 2 - Nested SYBR real time PCR & melt curves on (1) mini cultures, (2) amplicons from GoTaq


  • Part 1 - GoTaq "Dirty" PCR on liquid mini-cultures, 600 bp amplicons
    • Clone labeling notes, changed from last time:
      • L34 = Luc14 cells treated with gRNA034
      • G34 = Gal4EED/luc cells treated with gRNA034
      • 001, 002, 003 = clone no.
      • A01, A02, etc. = well position in dish or spot on agar array

1-96. L34_001_A01 - L34_096_H12 (full plate)
97-192. G34_001_A01 - G34_096_H12 (full plate)


Reagent Rxn1-24 Mix (x104) Expected:
PCR insert/ pJET = ~600 bp
1. L34_001_A01
2. L34_013_B01
3. L34_025_C01
4. L34_001_A01
Hover name
5 μL/lane, 1% agarose; Ladder
Template (culture) 2.0 ---
10 uM fwd primer 1.0 104.0
10 uM rev primer 1.0 104.0
2x GoTaq (clear) 12.5 1300.0
dH2O 8.5 884.0
  25.0 μL
  • Note: Master Mix Multiplier (for one 96-well plate) = 96 + 1 extra 8-well column = 104
  • Aliquot 200 μL into 8 tubes in 8-tube strip
  • Use multichannel to aliquot 23 μL to 96-well plate, 1 column at a time
  • Refill the 8-tube strip as needed with enough to fill remaining columns


PCR - Labnet: "GoTaq35KAH"

  • 95°C, 3 min
  • 35x[95°C, 10 sec; 60°C, 10 sec; 72°C, 20 sec]
  • 72°C, 3 min
  • 4°C ∞


CONCLUSIONS

  • PCR is not as efficient here as it was with the trial run (###)
  • Continue with other clones, but program step 1 as 98°C , 10 min.



Cas-tone - Phusion PCR for H3

  • Phusion PCR-amplify ORFs
  1. mEmerald-H3-23, H3_1 f1 / H3_40 r1
  2. mEmerald-H3-23, H3_1 f1 / H3_60 r1
  3. mEmerald-H3-23, H3_1 f1 / H3_80 r1
  4. mEmerald-H3-23, H3_1 f1 / H3_116 r1
  5. mEmerald-H3-23, H3_1 f1 / H3_136 r1
Reagent Vol Mix (x6) Expected:
1. H3-1-40 = 129
2. H3-1-60 = 189
3. H3-1-80 = 250
4. H3-1-116 = 373
5. H3-1-136 = 433
Hover name
5 μL/lane; 1% agarose; Ladder
Plasmid DNA 0.2 1.2
10 μM H3_1 f1 1.0 6.0
10 μM r primer 1.0 ---
10 mM dNTPs 1.0 6.0
5x HF buffer 10.0 60.0
Phusion Pol. 0.5 3.0
dH2O 36.3 217.8
  50.0 μL

Thermal cycler: Bio-Rad - Phusion

  • 98°C 3 min.
  • 30x[98°C, 10 sec; 70°C 30 sec; 72°C 30 sec]
  • 72°C 3 min.
  • 4°C, ∞