User:Karmella Haynes/Notebook/BioBrick cloning/2011/07/20

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07/20/11

  • ✓ Minipreps 1: KAH219, 223, 224/pSB31 (8 each)
  • ✓ Cultures/ minipreps 2: KAH213, 214, 215, 219, 220, 221, 222, 223, 224/MV1 (2 each)
  • ✓ Minipreps 3: High yield mp's for KAH220, 221, 222/pSB31 for Manching
  • ✓ Assemblies: miR-luc sensor "196/MV8 set"
  • ✓ Assemblies: Re-do KAH219, 223, 224/pSB31



Minipreps 1
> Check with KpnI/NcoI digests

Reagent Volume Expected:
pSB31 = 4500, 450

*pSB31 bands
**last frag + 300
1-8. 219/pSB31 5-8 = 4500*, 150*, 444, 1735, 532**
9-16. 223/pSB31 5-8 = 4500*, 150*, 636, 1190**
17-24. 224/pSB31 5-8 = 4500*, 150*, 636, 1490**
File:KAH 072011 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.


--> None worked. Re-do ligations.



Minipreps 2
> Check with E/P digests
> See 2/28/22 for example

Reagent Volume Expected:
Vector frag. = 4072
Insert + 1326...
1,2. KAH213/MV1 = 4160
3,4. KAH214/MV1 = 3083
5,6. KAH215/MV1 = 3383
7,8. KAH219/MV1 = 3748
9,10. KAH220/MV1 = 2671
11,12. KAH221/MV1 = 2971
13,14. KAH222/MV2 = 3940
15,16. KAH223/MV1 = 2863
17,18. KAH224/MV1 = 3163
File:KAH 072011 gel2.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.

--> Pick more colonies for 213, 219/MV1 (3 each)



Minipreps 3
> From 5 mL cultures from spot-streaks; combine duplicates
> Check with KpnI/NcoI digests

Reagent Volume Expected:
1. KAH220/pSB31 = 4500, 150, 444, 1190
2. KAH221/pSB31 = 4500, 150, 444, 1490
3. KAH222/pSB31 = 4500, 150, 636, 1735
See 7/18/11
File:KAH 072011 gel3.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.

> Measure conc.'s

Sample OD260 260/280 ng/μL
1. KAH220/pSB31 mp 2.229 1.86 111.4
2. KAH221/pSB31 mp 0.893 1.92 44.7
3. KAH222/pSB31 mp 0.893 1.92 44.7

--> Target concentration is 500 ng/μL. Concentrations too low. Zymo clean & concentrate tomorrow. Re-do digest.



Assemblies
--> Last time (6/06/11), may have used too much insert. Use calculated amount this time
--> Re-do digest of 196/MV8 and 198/MV8
--> Today, do 196/MV8 ligations

  1. KAH186-196/MV8: KAH186/(E/S)/2025 ✓ + (1) KAH196/MV8(E/X)/7978
  2. KAH184-196/MV8: KAH187/(E/S)/2269 ✓ + "
  3. KAH187-196/MV8: KAH187/(E/S)/2269 ✓ + "
  4. KAH188-196/MV8: KAH188/(E/S)/2124 ✓ + "
  5. KAH189-196/MV8: KAH189/(E/S)/2296 ✓ + "
  6. KAH190-196/MV8: KAH190/(E/S)/2368 ✓ + "
  7. KAH191-196/MV8: KAH191/(E/S)/2223 ✓ + "
  8. KAH183-196/MV8: KAH191/(E/S)/2223 ✓ + "
  9. KAH192-196/MV8: KAH192/(E/S)/2461 ✓ + "

(2) KAH198/MV8(E/X)/8050 (vector for next set)


> Digests (Fermentas FD)

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. KAH196/MV8 (E/X) 0.711 1.87 35.6
2. KAH198/MV8 (E/X) 0.893 1.92 44.7


> Ligations

Ligation Plate results (lig : neg crtl) 07/14/11
1-9. KAH###(E/S)/~2200, 11 ng + KAH196/MV8(E/X)/7978, 20 ng Notes: #:1 for all plates
10. KAH196/MV8(E/X)/ 20 ng  
  1 2 3 4 5 6 7 8 9 10
Insert DNA 1.0 1.0 1.5 1.0 1.0 0.5 1.0 0.5 0.8 ---
Vector DNA 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6
2x lgn buf (Roche) 5.0 5.0 5.0 5.0 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 1.0 1.0 1.0 1.0
dH2O 2.4 2.4 1.9 2.4 2.4 2.9 2.4 2.9 2.6 3.4
  10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL

--> Add to 30 μL DH5α Turbo; plate on 100 μg/mL amp



Assemblies
> Repeat from 7/17/11
> Use max vol. insert for 12 μL rxns.

  1. KAH219/pSB31: KAH219/(E/S blunt)/2422 ✓ + pSB31/(E blunt dp)/5127 ✓
  2. KAH223/pSB31: KAH223/(E/S blunt)/1537 ✓ + "
  3. KAH224/pSB31: KAH224/(E/S blunt)/1837 ✓ + "


> Ligations

Ligation Plate results (lig : neg crtl) 01/23/11
1. KAH219(E/S blunt)/2422, 24 ng + pSB31(E blunt dp)/5127, 25 ng KAH219/pSB31 2:1 (Pick 4)
5. KAH223(E/S blunt)/1537, 15 ng + " KAH223/pSB31 2:1 (Pick 4)
6. KAH224(E/S blunt)/1837, 18 ng + " KAH224/pSB31 5:1 (Pick 4)
7. pSB31(E blunt dp)/ 25 ng  
  1 2 3 4
Insert DNA 4.0 4.0 4.0 ---
Vector DNA 1.0 1.0 1.0 1.0
2x lgn buf (Roche) 6.0 6.0 6.0 6.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0
dH2O --- --- --- 4.0
  10 μL 10 μL 10 μL 10 μL

--> ~1 hr/ r.t.; Add to 30 μL DH5α turbo