User:Karmella Haynes/Notebook/BioBrick cloning/2010/01/20

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01/20/10

  • ✓ PCR: KAH94, 95/JDS48/49 minipreps from 1/19
  • ✓ Minipreps: KAH139, 140, 141 (2 each); KAH118/MV2(12)
  • ✓ Assemblies: KAH139, 140, 141/ V0201

Minipreps
> Check with E/P digests

Reagent 1-6 7-18 Expected:
V0120 = 3200
MV2 = 4529
1,2. KAH139 = 2945
3,4. KAH140 = 2939
5,6. KAH141 = 2527
7-18. KAH118/MV2 = 7930
E/P digests 1/20/10
15 μL/lane; 1% agarose
DNA(plasmid) 2.0 4.0
10X buffer 1.5 1.5
EcoRI 1.0 1.0
PstI 1.0 1.0
dH2O 9.5 7.5
  15 μL 15 μL --> 37°C/ ~15 min.

PCR
--> Check KAH94, 95/JDS48/49 minipreps from 1/19 (24 total)
--> Primers: RFP1 (used for RFP expression RT-PCR; should also work on DNA)

Reagent Volume   Expected:
1-6. KAH94/JDS49 = 100
7-12. KAH95/JDS49 = 100
13-18. KAH94/JDS48 = 100
19-24. KAH95/JDS48 = 100
25. KAH94/MV2 = 100
26. MV5 (NLS-pcDNA) = 0
PCR 1/20/10
10 μL/lane, 1% agarose
DNA (plasmid) 0.5
10 μM primer mix 1.0
2x GoTaq Green 10.0
dH2O 8.5
  20 μL

--> PCR (96-well): 95°C/ 3 min.; [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec] x30; 72°C/ 3 min.
--> Still unclear. Tomorrow digest w/ BglII


Assemblies

  1. KAH139/V0201: (1) KAH139/(N/S)/2945 + (4) V0201/(N/S)/5157
  2. KAH140/V0201: (2) KAH140/(N/S)/2939 + "
  3. KAH141/V0201: (3) KAH141/(N/S)/2527 + "

> Digests (Fermentas FD)
--> Use ScaI to cut V0120 for KAH139, 140

Reagent 1,2 1,2 Cloning digests 1/20/10
30 μL/lane, 1% agarose
DNA (plasmid) 10.0 10.0
10x buffer 3.0 3.0
NotI 1.0 1.0
SpeI 1.0 1.0
ScaI 1.0 ---
dH2O 15.0 15.0
  31 μL 30 μL --> 37°C/ ~30 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. KAH139(N/S) 0.350 2.03 17.5
2. KAH140(N/S) 0.443 1.81 22.2
3. KAH141(N/S) 0.451 2.02 22.5
4. V0201(N/S) 0.536 1.87 26.8


> Ligations

Ligation Plate results (lig : neg crtl) 01/21/10
1. KAH139(N/S)/2945, 29 ng + V0201(N/S)/5127, 25 ng KAH139/V0201 10:1 (Pick 2)
2. KAH140(N/S)/2939, 29 ng + " KAH140/V0201 10:1 (Pick 2)
3. KAH141(N/S)/2527, 25 ng + " KAH141/V0201 10:1 (Pick 2)
4. V0201(N/S)/ 25 ng  
  1 2 3 4
Insert DNA 2.0 2.0 2.0 ---
Vector DNA 1.0 1.0 1.0 1.0
2x lgn buf (Roche) 5.0 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0
dH2O 1.0 1.0 1.0 3.0
  10μL 10μL 10μL 10μL --> R.T./ 10 min.; Add 20 μL Z-DH5α



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