User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/22

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04/22/15

  • Rene - minipreps & sequencing: g53-2, g54
  • LCR Development - assembly attempt #1
  • Ryan - streak plates & liquid cultures (see 4/21/15)



Rene - minipreps & sequencing

  1. gRNA53-2
  2. gRNA54
  • Sigma GenElute kit
  • Elute w/ 75 μL elution sln.
  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. g53-2 0.186 1.863 186.1
2. g54 0.205 1.895 205.2


  • Submit to DNASU for sequencing
    • Order Number is: 10068 (4/23/15)
  1. g53-2, f (P139) - Confirmed
  2. g53-2, r (P139) - low Phred 20 score
  3. g54, f (P139) - Confirmed
  4. g54, r (P139) - low Phred 20 score



LCR - assembly attempt #1


Column-purify PCR fragments

  • Use Qiagen PCR Clean-up, elute and back-elute w/ 30 μL elution buffer
  1. Gal4DB-mCh (4/22/15)
  2. ATF2 (4/22/15)
  3. MV10 (4/17/15)


  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. 5'p Gal4DB-mCh PCR 0.139 1.825 138.6
2. 5'p ATF2 PCR 0.191 1.828 190.8
2. 5'p MV10 PCR 0.066 1.716 65.9


Dilute the purified dsDNA to 30 fmol/μL (30 nM)

  • Formula: x μL = length in bp ÷ measured ng/μL * 0.0195 ng/μL * 50μL
    • Gal4DB-mCh (1170 bp) = 8.2
    • ATF2 (906 bp) = 4.6
    • MV10 (5191 bp) = 76.8 -- use 19.2 to make 25 uL of 15 fmol/μL sln., use 2x
  • Add x to dH2O, final vol. = 50 μL
  • Use 1.0 μL of diluted dsDNA per 10 μL LCR reaction


Prep the Oligo Bridges

  • Note: Final conc. in LCR rxn. is 30 nM each
  • Bring the IDT oligo pellet to 100μM with dH2O. nmoles oligo (on label) * 10 = μL H2O to add
  • Make a 300 nM working solution (final volume = 100 μL) in a new tube. 3 μL of 100μM oligo stock + 97 μL dH2O = 100 μL


LCR Reactions (PCR fragments / oligo bridges)

  1. Gal4DB-mCh, ATF2, MV10 / LCRb_MV10_Gal4DB_rc, LCRb_mCh_ATF2_rc, LCRb_ATF2_MV10_rc
  2. Neg: MV10 / LCRb_MV10_Gal4DB_rc, LCRb_mCh_ATF2_rc, LCRb_ATF2_MV10_rc
  • Note: use 5.0 μL of 15 fmol/μL MV10 instead of 2.5
Reagent Rxn1 Rxn2 (neg)
30 nM DNA (3 nM) 10.0 5.0
300 nM Oligo Bridges (30 nM) 7.5 7.5
10X Ampligase Buffer 2.5 2.5
Ampligase 1.0 1.0
dH2O 4.0 6.5
  25.0

Thermocycler: BioRad dual block

  • 94 °C, 2 min.
  • 50x[94°C, 10 sec; 55°C, 30 sec; 66°C, 60 sec
  • 4 °C, ∞


Transformation

  • Add to 60 μL DH5α-turbo; incubate 5 min/ice
  • Plate on 100 μg/mL amp
  • Grow at 37°C overnight



Ryan - Receiver cloning confirmations

  • Check with E/X digests
Reagent Volume Expected:
1,2. AubR/MRV = ~3200, 886
3,4. BjaR/MRV = ~3200, 776
5,6. BraR/MRV = ~3200, 776
7,8. RpaR/MRV = ~3200, 779
MRV (empty) = ~3200
Hover name
15 μL/lane; 1% agarose; Ladder
DNA(plasmid) 3.0 μL
10X buffer 1.5
EcoRI 1.0
XbaI 1.0
dH2O 8.5
  15 μL --> 37°C/ ~15 min.


Conclusions

  • AubR/MRV - success! Use clone 1 (lane 1)
  • BjaR/MRV - success! Use clone 1 (lane 3)
  • BraR/MRV - failed. Pick more clones
  • RpaR/MRV - success! Use clone 2 (lane 8)



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