User:Karmella Haynes/Notebook/BioBrick cloning/2015/06/23

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06/23/15

  • Cas-tone - Assemblies: H3 parts into MV11
  • Rene - CRISPR library, continue dirty PCR



Cas-tone Assemblies

  • Do-over - use inserts with improved reverse primer (eliminate frame-shift)
  1. H3-1-40_MV11: H3-1-40/(X/N)/129 + MV11/(S/N)/9506
  2. H3-1-60_MV11: H3-1-60/(X/N)/189 + "
  3. H3-1-80_MV11: H3-1-80/(X/N)/250 + "
  4. H3-1-116_MV11: H3-1-116/(X/N)/373 + "
  5. H3-1-136_MV11: H3-1-136/(X/N)/433 + "


  • Digest of vector (Fermentas FD)
  1. MV11 - SpeI/NotI
Reagent Volume Hover name
30 μL/lane, 1% agarose; Ladder
DNA (plasmid) 20.0
10x buffer 3.0
SpeI 1.0
NotI 1.0
dH2O 5.0
  30 μL --> 37°C/ ~15 min.
  • Gel purification
    • Sigma GenElute kit. Elute & back elute w/ 25 μL elution sln.


  • DpnI digest & clean-up of PCR products (inserts)
    • Add 1 μL DpnI to each PCR rxn. Incubate @ 37°C/ 5 min.
    • Use the Qiagen Qiaquick PCR Purification kit (cat. no. 28104 ) to purify DNA.
    • Elute & back-elute with 30 μL of elution solution.


  • Measure [DNA] for vector and inserts
Sample OD260 260/280 ng/μL
1. MV11 (S/N) 0.019 1.596 19.4
2. H3-1-40 PCR 0.024 1.908 24.2
3. H3-1-60 PCR 0.041 1.984 40.8
4. H3-1-80 PCR 0.044 1.917 43.8
5. H3-1-116 PCR 0.067 1.865 67.1
6. H3-1-136 PCR 0.072 1.901 72.4


  • Digest & dephos inserts
    • XbaI/NotI
Reagent Volume
DNA (500 ng) up to 15.0 μL
10X FD buffer 2.0
FD XbaI 1.0
FD NotI 1.0
Roche SAP 1.0
dH2O x μL
  20.0

Thermal cycler: LabNet OptiMax "AnOlig shrt"

  • 37°C, 10 min
  • 95°C, 5 min
  • Ramp down to 25°C, 5°C/1 min. [90/1 min, 85/1 min, 80/1 min, ... 25°C/1 min]
  • 25°C, ∞
  • Final concentration = 25 ng/μL.


  • Ligations
    • > 2:1 ratio calculations...
    • 433 bp largest insert / 9506 bp vector * 2 * 50 ng vector = 4.5 ng
    • 129 bp smallest insert / 9506 bp vector * 2 * 50 ng vector = 1.4 ng
Reagent Rxn1-5 Rxn6
Insert DNA 0.5 ---
Vector DNA 2.5 2.5
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O 1.0 1.5
  10.0 μL 10.0 μL

RESULTS (6/24/15)

  • SUCCESS! Plate 6 (neg ctrl) has 7 colonies; Plates 1-5 have 30 - 50 colonies
  • Pick two clones from each for 5 mL cultures/ minipreps



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_097_A01 - L34_192_H12 (full plate)


Reagent Rxn1-24 Mix (x104) Expected:
PCR insert/ pJET = ~600 bp
1. L34_097_A01
2. L34_109_B01
3. L34_121_C01
4. L34_133_D01
5. L34_145_E01
6. L34_157_F01
7. L34_169_G01
8. L34_181_H01
Gel showed no signal
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"

  • 98°C, 10 min
  • 35x[95°C, 10 sec; 60°C, 10 sec; 72°C, 20 sec]
  • 72°C, 3 min
  • 4°C ∞


CONCLUSIONS

  • Looks like amplification failed
  • Move on to sequencing of all clones
  • Restart fresh cultures tomorrow
  • Will submit as pellets to DNASU