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

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04/16/15

  • LCR Development - PCR MV10 bacbone
  • 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 Rxn1,2 Rxn3,4 Expected:
1,2. Gal4DB-mCh = 1170
3,4. ATF2 = 906
 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

Program: Phusion (block B)

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


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. ###/###
  2. ###/###
  3. ###/###


Reagent Rxn
100 ng Vector ##
1:250 dsOligo 2.0
10x FD buf (NEB) 2.0
10 mM DTT 1.0
10 mM ATP 1.0
FastDigest BbsI 1.0
T7 DNA ligase 0.5
dH2O ##
  20.0

Bio-Rad Thermocycler

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


Transformation(s)

  • 2.0 μL ligation + 50 μL DH5α-turbo
  • Plate on 100 μg/mL amp




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