User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/26

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05/26/15

  • Ben - luc replacement donor plasmid DBN007
  • Rene - CRISPR PCR library PCR trial



Ben - luc replacement donor plasmid

  • Assembly:
  1. DBN007_pSB1A3: DBN006(BsaI)/1972 + DBN001(BsaI)/1267+1069 (=2336)


Reagent Rxn1,2 Expected:
1,2. DBN006 = 1972
Hover name
5 μL/lane, 1% agarose; Ladder
Template 0.2
10 uM fwd primer 1.0
10 uM rev primer 1.0
10 mM dNTPs 1.0
Phusion pol. 0.5
5x HF buffer 5.0
dH2O 41.3
  50.0

Bio-Rad: "Phusion" (block #)

  • 98°C, 3 min
  • 30x[98°C, 10 sec; 67°C, 30 sec; 72°C, 30 sec]
  • 72°C, 10 min
  • 4°C ∞

CONCLUSION

  • Non-specific amplification. Need to gel purify.
  • Use 30 uL of each per lane for gel purification.


CONTINUED: 5/27/15


  • Gel purification
    • Use Sigma kit
    • Elute & back-elute w/ 25 μL elution sln.
Sample OD260 260/280 ng/μL
1. DBN006 PCR 0.002 -19 2.0
  • Concentration is weird. Use max amt. of DNA for subsequent steps


  • Digest & Dephosphorylate the Insert(s)
    • Use BsaI
Reagent Volume
DNA (500 ng) up to 16.0 μL
10X FD buffer 2.0
FD BsaI 1.0
Roche SAP 1.0
dH2O x μL
  20.0

Thermal cycler program: Note: In the Haynes lab, use the LabNet OptiMax Thermocycler, Program "AnOlig RD"

  • 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, ∞
  • The final concentration is ???


  • Ligations
    • DBN001 2-part backbone from 05/15/15
    • 2:1 ratio calc.: 1972 bp insert / 2336 bp vector * 2 * 50 = 84 ng insert
  1. DBN006/(BsaI)/1972 + DBN001/(BsaI)/2336
  2. DBN001/(BsaI)/2336
Reagent Rxn1 Rxn2
Insert DNA 6.0 ---
Vector DNA 3.0 3.0
2x lgn buf (Roche) 10.0 10.0
T4 ligase (NEB) 1.0 1.0
dH2O --- 6.0
  20.0 μL 20.0 μL
  • Incubate at RT/ 10 min.
  • Add to 50 μL DH5α-turbo; ice/ 5min.
  • Plate on 100 μg/mL amp agar


RESULTS (5/28/15)

  • Success! 50 colonies on ligation plate, 5 colonies on neg. ctrl. Pick two colonies



Rene - CRISPR PCR library PCR trial

  • 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:
      • Lu34 = Luc14 cells treated with gRNA034
      • Ga34 = Gal4EED/luc cells treated with gRNA034
      • A/B = 96-well plate A or B
      • A01, A02, etc. = well position in dish or spot on agar array

1-12. Luc14 g034 - Lu34_AA01 - Lu34_AA12
13-24. Gal4EED g034 Ga34_AA1 - GaA_12


Reagent Rxn1-24 Mix (x25) Expected:
1-14. PCR insert/ pJET = ~600 bp
Hover name
10 μL/lane, 1% agarose; Ladder
Template (culture) 0.5 ---
10 uM fwd primer 1.0 25.0
10 uM rev primer 1.0 25.0
2x GoTaq (clear) 12.5 312.5
dH2O 10.0 250.0
  25.0 μL

Labnet: "GoTaq"

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


CONCLUSIONS

  • Success with dirty PCR
  • Continue with remainder of dirty PCR & possible SYBR PCR tomorrow




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