User:Karmella Haynes/Notebook/BioBrick cloning/2012/07/27

From OpenWetWare

Jump to: navigation, search
Karmella's BioBrick Cloning Main project page
Previous entry      Next entry

07/27/12

  • Assembly: clone linker peptides into V0120
  • DNA sequencing order: order M13F primer sequencing fro FCt2 plasmid online (DNASU)



Assemblies
> Repeat cloning from 6-27-12

  1. PLrigid/V0120: ✓ PLrigid/(X/P)/60 + V0120/(X/P)/~3200
  2. PLrigid4/V0120: ✓ PLrigid4/(X/P)/240 + "
  3. PLflex/V0120: ✓ PLflex/(X/P)/60 + "
  4. PLflex4/V0120: ✓ PLflex4/(X/P)/240 + "


> Digests (Fermentas FD)
--> Digest V0120 backbone only. Inserts are already done.

Reagent Volume  
DNA (plasmid) 20.0
10x buffer 3.0
XbaI 1.0
PstI 1.0
dH2O 5.0
  30 μL --> 37°C/ ~30 min.

---> Zymo clean; elute w/ 15 μL dH2O


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. V0120 (X/P) 0.009 2.049 8.9


> Ligations
--> 60 bp inserts = 1/50 total mass; 240 bp inserts are 1/12 of total mass; multiply ng used accordingly
--> Use V0120 from ccdB
--> Use dephos insert only as neg ctrls.

Ligation Plate results (lig : neg crtl) 07/28/12
1. PLrigid(X/P)/60, 1 ng (x50) + V0120(X/P)/3200, 25 ng PLrigid/V0120 1:1 (try again)
2. PLrigid(X/P)/60, 1 ng (x50) + no vector ~10
3. PLrigid4(X/P)/240, 4 ng (x12) + V0120(X/P)/3200, 25 ng PLrigid4/V0120 1:1 (try again)
4. PLrigid4(X/P)/240, 4 ng (x12) + no vector ~10
5. PLflex(X/P)/60, 1 ng (x50) + V0120(X/P)/3200, 25 ng PLflex/V0120 1:1 (try again)
6. PLflex(X/P)/60, 1 ng (x50) + no insert ~10
7. PLflex4(X/P)/240, 4 ng (x12) + V0120(X/P)/3200, 25 ng PLrigid4/V0120 1:1 (try again)
8. PLflex4(X/P)/240, 4 ng (x12) + no insert ~10
  1 2 3 4 5 6 7 8
Insert DNA 2.2 2.2 2.2 2.2 2.2 2.2 2.2 2.2
Vector DNA 2.8 --- 2.8 --- 2.8 --- 2.8 ---
2x lgn buf (Roche) 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
dH2O --- 2.8 --- 2.8 --- 2.8 --- 2.8
  12 μL 12 μL 12 μL 12 μL 12 μL 12 μL 12 μL 12 μL

--> Add 30 μL DH5α Turbo cells; plate on 100 μg/mL Amp


Personal tools