User:Karmella Haynes/Notebook/BioBrick cloning/2011/01/25

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01/25/11

  • ✓ Minipreps: KAH160/pSB31 (10)
  • ✓ Cultures & minipreps: KAH165/pSB31 (10); 2 mL
  • ✓ Assemblies: KAH179, 180 (CFP-Targets) into KAH176, 177, 178/MV8 (RFP-Repressor expression vectors); continue later
  • ✓ Cultures: SP1AB 3x mut (24); 2 mL



Minipreps
> Check with KpnI/NcoI digests
--> pSB31 KpnI is located 142 bp upstream of EcoRI cloning site (according to pdf map)
--> BioBrick constructs have 2 internal NcoI sites

Reagent Volume Expected (approx.):
1,2. KAH170pSB31 2,5
fwd = 5500, 444, 150
rev = 5010, 640, 444
3-12. KAH160 7-16
fwd = 5500, 636, 150
rev = 5010, 640, 636

Bottom
1-10. KAH165 7-16 = 636, 5480
1/25/11 Miniprep digests
15 μL/lane; 1% agarose

1/25/11 Miniprep digests #2
KAH165/pSB31 minipreps, NcoI only
DNA(plasmid) 2.0 μL
10X buffer 1.5
KpnI 1.0
NcoI 1.0
dH2O 9.5
  15 μL --> 37°C/ ~15 min.

--> Conclusions: KpnI digestion unclear; successfully identified KAH160/pSB31 clone; continue w/ picking more KAH165/pSB31 clones before sending plasmids in for sequencing.
--> ID'ed two promising KAH165/pSB31 clones; send five putatives off for sequencing (1/26/11 Snow emergency: Genewiz not picking up orders until further notice)



Assemblies

  1. KAH179/176/MV8: (1) KAH179/(E/S)/2941 + (3) KAH176/MV8/(E/X)/7785
  2. KAH180/176/MV8: (2) KAH180/(E/S)/2743 + "
  3. KAH179/177/MV8: KAH179/(E/S)/2941 + (4) KAH177/MV8/(E/X)/7978
  4. KAH180/177/MV8: KAH180/(E/S)/2743 + "
  5. KAH179/178/MV8: KAH179/(E/S)/2941 + (5) KAH178/MV8/(E/X)/7989
  6. KAH180/178/MV8: KAH180/(E/S)/2743 + "


> Digests (Fermentas FD)
--> Note: Re-digested KAH179 w/ E/S/ApaLI

Reagent Volume   01/25/11 Cloning digest
30 μL/lane, 1% agarose
DNA (plasmid) up to 25 μL
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O ---
  30 μL --> 37°C/ ~30 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. KAH179 (E/S) 0.167 2.32 8.4
2. KAH180 (E/S) 0.109 3.19 5.5
3. KAH176/MV8 (E/X) 0.407 2.02 20.3
4. KAH177/MV8 (E/X) 0.441 2.22 22.0
5. KAH178/MV8 (E/X) 0.164 2.69 8.2


--> Continue with ligation later


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