User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/28

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05/28/15

  • David Tze - assemblies: linkers + hPCD
  • Ben - DBN007 5 mL cultures



David Tze - assemblies: linkers + hPCD

  • Assemblies
  1. DT001_pUC57: hPCD(E/S)/186 + PLflex_pUC57(E/X)/2763
  2. DT002_pUC57: hPCD(E/S)/186 + PLflex4_pUC57(E/X)/2930
  3. DT003_pUC57: hPCD(E/S)/186 + PLrigid_pUC57(E/X)/2763
  4. DT004_pUC57: hPCD(E/S)/186 + PLrigid4_pUC57(E/X)/2931


  • Digests (Fermentas FD)
Reagent Rxn1 Rxn2-5 Expected bands:
1. hPCD(E/S) = 3200, 186*
2. PLflex_pUC57(E/X) = 2763*
3. PLflex4_pUC57(E/X) = 2930*
4. PLrigid_pUC57(E/X) = 2763*
5. PLrigid4_pUC57(E/X) = 2931*

(*) Bands that were cut out for purification
Hover name
30 μL/lane, 1% agarose; Ladder
DNA (plasmid) 25.0 15.0
10x buffer 3.0 3.0
EcoRI 1.0 1.0
enzyme 2 1.0 1.0
dH2O --- 10.0
  30 μL

--> 37°C/ ~30 min.


  • Gel purification
    • Sigma GenElute Gel purification kit
    • Elute & back-elute w/ 25 μL elution sln.


  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. hPCD(E/S) --- --- ---
2. PLflex_pUC57(E/X) --- --- ---
3. PLflex4_pUC57(E/X) --- --- ---
4. PLrigid_pUC57(E/X) --- --- ---
5. PLrigid4_pUC57(E/X) --- --- ---
    • Readings are very odd, which is typical for the Sigma gel extraction products.
    • Will try my luck with these and use an arbitrary volume of 2.0 insert and 2.0 vector.
    • Good insert : vector ratio should not be difficult to achieve since insert is very short.


  • Ligations
    • 2:1 ratio calculation: 186 bp insert / ~2800 bp vector * 2 * 50 = 6.64 ng insert
  1. DT001_pUC57: hPCD(E/S)/186 + PLflex_pUC57(E/X)/2763
  2. DT002_pUC57: hPCD(E/S)/186 + PLflex4_pUC57(E/X)/2930
  3. DT003_pUC57: hPCD(E/S)/186 + PLrigid_pUC57(E/X)/2763
  4. DT004_pUC57: hPCD(E/S)/186 + PLrigid4_pUC57(E/X)/2931
  5. PLflex_pUC57(E/X)/2763
  • Note: not enough plates to do neg. ctrls for all vectors. Used just one of the vectors
Reagent Rxn1-4 Rxn5
Insert DNA 2.0 ---
Vector DNA 2.0 2.0
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O --- 2.0
  10.0 μL 10.0 μL


RESULTS

  • Looks successful - David Tze: ~200 colonies on the ligation plates, 1 colony on the neg. ctrl plate.
  • David Tze: picked two colonies from each plate (1-4) for streak library and 5 mL cultures



Ben - DBN007 5 mL cultures


  • Minipreps
    • Sigma GenElute kit, elute w/ 75 μL elution sln.


  • Digests
    • Cut w/ E/S
Reagent Volume Expected:
1-2. DBN007_pSB1A3 = 2197, 2132
DBN001 = 2132, 224
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/ ~15 min.


CONCLUSIONS

  • Looks successful. 2197, 2132 bands are hard to resolve at the 2000 kb range.
  • Submit the plasmids for sequencing