User:Karmella Haynes/Notebook/BioBrick cloning/2011/07/04

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07/04/11

  • Assemblies 1: KAH201, 202, 203 ✓
  • Assemblies 2: Redo miR sensor assemblies (sub-set #1) (on hold: order more ligase)
  • Re-transformation: 5xGal4 KMC01 ✓

-->Note: incubate plates at 37C for 3 hours, put on bench overnight



Assemblies 1

  1. KAH201: 5xGal4 KMC01/(S/P)/93 ✓ + (1) KAH199/(X/P dp)/127
  2. KAH202: " + (2) KAH199/(X/P dp)/127
  3. KAH203: (3) 5xGal4 KMC01/(E/S dp)/93 + HSVtk TATA DB81/(X/P)/56 ✓

> Digests (Fermentas FD)
--> Specific notes

Reagent Volume  
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.


> Zymo clean
--> Elute with 25 μL dH2O


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. KAH199 (X/P) --- --- ---
2. KAH200 (X/P) --- --- ---
3. 5xGal4 KMC01 (E/S) --- --- ---


> Dephosphorylation (Roche)

Reagent Volume
DNA (clean digest) up to 17 μL (500 ng)
10x buffer d.p. 2.0
phosphatase 1.0
dH2O ---
  20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL


> Ligations

Ligation Plate results (lig : neg crtl) mm/dd/yy
1. KAH199(X/P dp)/127, 2 ng + 5xGal4 KMC01(S/P)/~3293, 25 ng KAH201 >10:1 (Pick 3)
2. KAH200(X/P dp)/127, 2 ng + " KAH202 >10:1 (Pick 3)
3. 5xGal4 KMC01(S/P)/ 25 ng  
4. 5xGal4 KMC01(E/S dp)/93, 1 ng + HSVtk TATA DB81(E/X)/~3256, 25 ng KAH203 >10:1 (Pick 3)
5. HSVtk TATA DB81(E/X)/ 25 ng  
  1 2 3 4 5
Insert DNA 3.6 3.6 --- 3.7 ---
Vector DNA 0.4 0.4 0.4 0.3 0.3
2x lgn buf (Roche) 5.0 5.0 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0
dH2O --- --- 3.6 --- 3.7
  10 μL 10 μL 10 μL 10 μL 10 μL

--> Add rxn. to 30 μL DH5α Turbo cells



Assemblies 2
--> Sequencing failed twice for miR sensor assemblies. Daniel tried ApaLI digest on a couple and saw no cutting (in previous digests, EcoRI probably cut the "vector"). Plasmids are strange. Re-do ligations, but check with ApaLI only from now on.
--> Re-do miR sensor set 1, see 6/06/11

> Ligations

Ligation Plate results (lig : neg crtl) 07/05/11
1-9. KAH###(E/S)/~2200, 11 ng + KAH196(E/X)/7806, 20 ng Notes: >10:1 for all plates
10. KAH196(E/X)/ 25 ng  


--> Note: Dilute vector to 20 ng/μL before use
--> Use 2:1 ratio of insert, do not max out volume

  1-9 10
Insert DNA 3.0 ---
Vector DNA 0.5 0.5
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O 0.75 3.75
  10 μL 10 μL

--> Add rxn. to 25 μL DH5α Turbo cells