User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/16

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04/16/15

  • LCR Development - PCR MV10 backbone
  • gRNA cloning (Rene)



LCR Development

  • Try PCR on linearized vector
Reagent Volume
DNA(plasmid) 5.0 μL
10X buffer 3.0
XbaI 2.0
dH2O 20.0
  30 μL --> 37°C/ ~30 min.


Measure conc.

  • DNA purified with Sigma PCR clean-up kit
  • Eluted& back-eluted with 30 μL elution sln.
Sample OD260 260/280 ng/μL
1. MV10 XbaI 0.017 1.47 17.24


Phusion PCR

  1. 1.0 μL template, HF buffer
  2. 2.0 μL template, HF buffer
  3. 1.0 μL template, GC buffer
  4. 2.0 μL template, GC buffer
Reagent Rxn1,3 Rxn2,4 Expected:
MV10 band = 5191
Hover name
10 μL/lane, 1% agarose; Ladder
Template 1.0 2.0
10 uM fwd primer 1.0 1.0
10 uM rev primer 1.0 1.0
10 mM dNTPs 1.0 1.0
Phusion pol. 0.5 0.5
5x buffer (HF/GC) 5.0 5.0
dH2O 40.5 39.5
  50.0 50.0

Mistake: Buffer volume should be 10/rxn, no 5! Consider re-run

Program: Phusion (block B)

  • 98°C, 3 min
  • 35x[98°C, 10 sec; 60°C, 30 sec; 72°C, 2.5 min]
  • 72°C, 10 min
  • 4°C ∞

Conclusion

  • Not ideal, proceed with caution


Measure conc.

  • PCR 1 & 2 combined, purified with Sigma PCR clean-up kit
  • Eluted& back-eluted with 30 μL elution sln.
Sample OD260 260/280 ng/μL
1. 5'p-MV10 PCR 0.008 1.28 7.77



gRNA cloning - Rene

  • Follow the "Oligo annealing and cloning into backbone vectors - NEW" protocol

Phosphorylate and anneal oligo pairs

  1. g051 T/B
  2. g052 T/B
  3. g053 T/B
  4. g054 T/B
  5. g055 T/B
  6. g056 T/B
Reagent Rxn Mix (7x)
100 μM Oligo 1 1.0 ---
100 μM Oligo 2 1.0 ---
10x T4 Lign buf (NEB) 1.0 7.0
T4 PNK (NEB) 0.5 3.5
dH2O 6.5 45.5
  10.0


LabNet OptiMax Thermocycler: AnOlig RD

  • 37°C, 30 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, ∞


Dilute the product(s) 1:250

  • Add 2 μL product to 498 μL dH2O


Digestion/ Ligation Reactions (Vector / dsOligo Insert)

  1. pX330 / g051
  2. pX330 / g052
  3. pX330 / g053
  4. pX330 / g054
  5. pX330 / g055
  6. pX330 / g056


Reagent Rxn Mix (x7)
100 ng Vector 0.5 3.5
1:250 dsOligo 2.0 ---
10x FD buf 2.0 14.0
10 mM DTT 1.0 7.0
10 mM ATP 1.0 7.0
FastDigest BbsI 1.0 7.0
T4 DNA ligase 0.5 3.5
dH2O 12.0 84.0
  20.0

LabNet OptiMax Thermocycler: BbsI Dig/Lig

  • 6x [37°C, 5 min; 23°C, 5 min]
  • 4°C, ∞


Transformation(s)

  • Split ligations in 1/2, do rapid protocol for 1 set, long protocol for other set if needed
  • 10.0 μL ligation + 50 μL DH5α-turbo
  • Plate on 100 μg/mL amp


RESULTS (4/17/15)

  • Success! Plates 1-3, 5, 6 had ~5 colonies. Plate 4 had just one.
  • Pick two colonies from each (except plate 4, pick 1)
  • Make streak plate & set up 5 mL cultures




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