User:Karmella Haynes/Notebook/BioBrick cloning/2011/08/04

From OpenWetWare
Jump to: navigation, search
Owwnotebook icon.png Project name Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png

08/04/11

  • ✓ Minipreps: cultures from last night; KAH213/MV1 (4), KAH219/pSB31 (4), KAH223/pSB31 (4)
  • ✓ Assemblies: KAH234; KAH231, 232, 228/ MV1



Minipreps
1-4. Check with E/P
5-12. Check with KpnI/ NcoI

Reagent Volume Expected:
Vector frag. = 4072
Insert + 1326...
1-4. KAH213/MV1 = 4160

pSB31 (empty) = 4500, 450
*pSB31 bands
**last frag + 300
5-8. 219/pSB31 5-8 = 4500*, 150*, 444, 1735, 532**
9-12. 223/pSB31 5-8 = 4500*, 150*, 636, 1190**

File:KAH 080411 gel1.tif
15 μL/lane; 1% agarose
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 9.5
  15 μL --> 37°C/ ~15 min.

Assemblies

  1. KAH234: KAH201/(S/P)/203 ✓ + (1) KAH233/(X/P)/832
  2. KAH231/MV1: (2) KAH231/(E/S)/1588 + MV1/(E/X)/5428 ✓
  3. KAH232/MV1: (3) KAH232/(E/S)/1780 + "
  4. KAH228/MV1: (4) KAH228/(E/S)/2000 + "

> Digests (Fermentas FD)

Reagent Volume   File:KAH 080411 gel2.tif
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. KAH233 (X/P) 0.337 1.84 16.8
2. KAH231 (E/S) 0.892 1.87 44.6
3. KAH232 (E/S) 0.661 1.88 33.0
4. KAH228 (E/S) 0.239 1.66 11.9


> Ligations

Ligation Plate results (lig : neg crtl) mm/dd/yy
1. KAH233(X/P)/832, 10 ng + KAH201(S/P)/~3403, 20 ng KAH234 >10:1 (Pick 2)
2. KAH201(S/P)/ 20 ng  
3. KAH231(E/S)/1588, 12 ng + MV1(E/X)/5428, 20 ng KAH231/MV1 >10:1 (Pick 2)
4. KAH232(E/S)/1780, 13 ng + " KAH232/MV1 >10:1 (Pick 2)
5. KAH228(E/S)/2000, 15 ng + " KAH228/MV1 >10:1 (Pick 2)
6. MV1(E/X)/ 20 ng  
  1 2 3 4 5 6
Insert DNA 1.0 --- 0.5 0.5 2.0 ---
Vector DNA 3.0 3.0 0.5 0.5 0.5 0.5
2x lgn buf (Roche) 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
dH2O --- 1.0 3.0 3.0 1.5 3.5
  10 μL 10 μL 10 μL 10 μL 10 μL 10 μL