User:Karmella Haynes/Notebook/BioBrick cloning/2011/07/21

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07/21/11

  • ✓ Cultures/minipreps: miR-luc 196/MV8 set (9 constructs, 3 each); KAH219, 223, 224/pSB31 (4 each); KAH213, 219/MV1 (3 each)



Minipreps

  1. miR-luc 196/MV8 set: ApaLI
  2. KAH219, 223, 224/pSB31: KpnI/NcoI
  3. KAH213, 219/MV1: E/P
Reagent Volume Expected:
MV8 bands = 1243, 1135, 288
1-3. 186-196/MV8 = 3688*, 2772**, 711
4-6. 184-196/MV8 = 3854*, 2772**, 711
7-9. 187-196/MV8 = 3932*, 2772**, 711
10-12. 188-196/MV8 = 3787*, 2772**, 711
13-15. 189-196/MV8 = 3959*, 2772**, 711
16-18. 190-196/MV8 = 4031*, 2772**, 711
19-21. 191-196/MV8 = 3886*, 2772**, 711
22-24. 183-196/MV8 = 4046*, 2772**, 711
25-27. 192-196/MV8 = 4124*, 2772**, 711
*insert + 234 (vector) + 1429 (196 frag)
**last 196 frag + 1655



pSB31 = 4500, 450
28-31. 219/pSB31 = 4500*, 150*, 444, 1735, 532**
32-35. 223/pSB31 = 4500*, 150*, 636, 1190**
36-39. 224/pSB31 = 4500*, 150*, 636, 1490**
*pSB31 bands
**last frag + 300

MV1 frag. = 4072
40-42. 213/MV1 = 4160
43-45. 219/MV1 = 3748

Miniprep digests 1, 7/21/11
15 μL/lane; 1% agarose

Miniprep digests 2, 7/21/11
15 μL/lane; 1% agarose

Miniprep digests 3, 7/21/11
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. BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
  2. BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size

> 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.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. Digested part (a/b) --- --- ---
2. Digested part (c/d) --- --- ---


> 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. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng new BioBrick #:1 (Pick #)
2. vector(c/d)/ ## ng  
  1 2
Insert DNA ### ---
Vector DNA ### ###
2x lgn buf (Roche) ### ###
T4 ligase (NEB) 1.0 1.0
dH2O ### ###
  # μL # μL

Oligo annealing

  1. New BB 1
  2. New BB 2
DNA (oligos, 100 μM) up to 18 μL (3 μL ea.)
10x annealing buffer 2.0
dH2O ---
  20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight



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