User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/15

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05/15/15

  • Ryan - assembly (traditional) for Receiver plasmids
  • Ben - assembly for luc replacement
  • Cas-tone - minipreps & digests



Assemblies

  • For Ryan and Ben
  1. pAub-AubR_MRV: pAub-PCR(E/S dp)/153 + AubR/MRV(BbsI)/4087
  2. pBja-BjaR_MRV: pBja-PCR(E/S dp) + BjaR/MRV(BbsI)/3977
  3. pBra-BraR_MRV: pBra-PCR(E/S dp) + BraR/MRV(BbsI)/3977
  4. pRpa-RpaR_MRV: pRpa-PCR(E/S dp) + RpaR/MRV(BbsI)/3980
  5. DBN007_pSB1A3: DBN006(BsaI)/1972 + DBN001(BsaI)/1267/1069(2336)


  • Digests (Fermentas FD)
  1. AubR/MRV(BbsI)/4087
  2. BjaR/MRV(BbsI)/3977
  3. BraR/MRV(BbsI)/3977 (two bands, isolate both)
  4. RpaR/MRV(BbsI)/3980
  5. DBN006(BsaI)/1972 (this is in V0120...need PCR fragment)
  6. DBN001(BsaI)/1267/1069(2336) (two bands, isolate both)


Reagent Volume Hover name
30 μL/lane, 1% agarose; Ladder -->
DNA (plasmid) 15.0
10x buffer (FD) 3.0
enzyme 1 1.0
dH2O 11.0
  30 μL

--> 37°C/ ~30 min.


  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. AubR/MRV(BbsI) 0.051 1.924 51.4
2. BjaR/MRV(BbsI) 0.038 1.983 38.0
3. BraR/MRV(BbsI) 0.134 1.882 133.7
4. RpaR/MRV(BbsI) 0.047 1.858 46.7
5. DBN001 (BsaI) 0.017 2.176 16.8


  • Ligations ("traditional")
  1. pAub-PCR(E/S dp)/153 + AubR/MRV(BbsI)/4087
  2. AubR/MRV(BbsI)/4087
  3. pBja-PCR(E/S dp) + BjaR/MRV(BbsI)/3977
  4. BjaR/MRV(BbsI)/3977
  5. pBra-PCR(E/S dp) + BraR/MRV(BbsI)/3977
  6. BraR/MRV(BbsI)/3977
  7. pRpa-PCR(E/S dp) + RpaR/MRV(BbsI)/3980
  8. RpaR/MRV(BbsI)/3980
Reagent Rxn1 Rxn2 Rxn3 Rxn4 Rxn5 Rxn6 Rxn7 Rxn8
Insert DNA 2.0 --- 2.0 --- 2.0 --- 2.0 ---
Vector DNA 1.0 1.0 1.0 1.0 0.5 0.5 1.0 1.0
2x lgn buf (Roche) 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
dH2O 1.0 1.0 1.0 1.0 1.5 1.5 1.0 1.0
  10.0 μL 10.0 μL 10.0 μL 10.0 μL 10.0 μL 10.0 μL 10.0 μL 10.0 μL
  • Add 50 μL DH5α-turbo; ice 5 min.
  • Plate on 100 μg/mL amp
  • Grow overnight @37°C


RESULTS

  • Success! Ligation plates = ~50 colonies, Neg ctrls = 0 - 3 colonies



Cas-tone - minipreps, digests

  • Minipreps
    • Sigma GenElute mini kit; elute w/ 75μL elution sln.
    • Check with E/S digests - SpeI site (@250) is destroyed and new SpeI site (@844) is gained in successful ligation
  1. CMV-dCas9-VP64 1-1
  2. CMV-dCas9-VP64 1-2
  3. CMV-dCas9-VP64 2-1
  4. CMV-dCas9-VP64 2-2
Reagent Volume Expected:
1-4. CMV-dCas9-VP64 = ~5400, 3016, 1363
dCas9-VP64 = 4697, 3016, 2099
Hover name
15 μL/lane; 1% agarose; Ladder
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
SpeI 1.0
dH2O 9.5
  15 μL

--> 37°C/ ~10 min.


CONCLUSIONS

  • Success! Lane 4. Keep clone 2-2