User:Karmella Haynes/Notebook/BioBrick cloning/2009/12/29

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12/29/09

  • ✓ Minipreps: KAH136(2), 137(4), 138(4); retransformations KAH93(2), JDS42(2), JDS43(2)
  • ✓ Assemblies: KAH139, 140; KAH117-119; KAH94, 95/JDS42; KAH94, 95/JDS43
  • ✓ Cultures: KAH137, 138 (6 each)



Minipreps
> Check with E/P digests

Reagent Volume Expected:
1,2. KAH136 = 1941
3-6. KAH137 = 5058
7-10. KAH138 = 5130
11,12. KAH93 = 4839
13,14. JDS42 = ~1860, ~2050, ~3550
15,16. JDS43 = ~1860, ~2050, ~3550
Hover name
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. --> KAH137, 138 failed; pick 6 more colonies each


Assemblies

  1. KAH139: (1) KAH135/(E/S)/2353 + (3) NLSstop2(KAH011)/(E/X)/~3200
  2. KAH140: (2) KAH136/(E/S)/1941 + "
  3. KAH117: KAH99/(E/S)/2913 ✓ + (4) KAH93/(E/X)/8039
  4. KAH118: KAH100/(E/S)/7930 ✓ + "
  5. KAH119: KAH101/(E/S)/8002 ✓ + "
  6. KAH94/JDS42: KAH94/(E/P+Ad's)/5011 ✓ + (5) JDS42/(BglII)/7468
  7. KAH95/JDS42: KAH95/(E/P+Ad's)/5083 ✓ + "
  8. KAH94/JDS43: KAH94/(E/P+Ad's)/5011 ✓ + (6) JDS43/(BglII)/7478
  9. KAH95/JDS43: KAH95/(E/P+Ad's)/5083 ✓ + "

> Digests (Fermentas FD)
--> BglII single digest for JDS42, 43 (2.0 μL BglII)

Reagent Volume   Hover name
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. KAH135 (E/S) 0.669 2.19 33.5
2. KAH136 (E/S) 0.679 2.00 33.9
3. KAH010 (E/X) 0.500 2.37 25.0
4. KAH93 (E/X) 0.190 3.25 9.5
5. JDS42 (BglII) 0.313 2.42 15.7
6. JDS43 (BglII) 0.397 2.37 19.8


> Ligations
--> Use dsoligo adapters (Ad BglII/EcoRI and Ad PstI/BglII from 12/28/09) for JDS constructs

Ligation Plate results (lig : neg crtl) 12/30/09
1. KAH135(E/S)/2353, 29 ng + NLSstop2(E/X)/~3200, 20 ng KAH139 1:1 (re-clone)
2. KAH136(E/S)/1941, 24 ng + " KAH140 10:1 (re-clone)
3. NLSstop2(E/X)/ 20 ng lots of colonies
4. KAH99(E/S)/2913, 29 ng + KAH93(E/X)/8039, 40 ng KAH117 10:1 (Pick 2)
5. KAH100(E/S)/3085, 31 ng + " KAH118 10:1 (Pick 2)
6. KAH101(E/S)/3157, 32 ng + " KAH119 5:1 (Pick 4)
7. KAH93(E/X)/ 40 ng  
8. KAH94(E/P+Ad's)/5011, 40 ng + JDS42(BglII)/7468, 30 ng KAH94/JDS42 1:1 (re-clone)
9. KAH95(E/P+Ad's)/5083, 41 ng + " KAH95/JDS42 1:1 (re-clone)
10. JDS42(BglII)/ 30 ng very few colonies
11. KAH94(E/P+Ad's)/5011, 40 ng + JDS43(BglII)/7478, 30 ng KAH94/JDS43 3:1 (Pick 6)
12. KAH95(E/P+Ad's)/5083, 41 ng + " KAH95/JDS43 3:1 (Pick 6)
13. JDS43(BglII)/ 30 ng very few colonies
  1 2 3 4 5 6 7 8 9 10 11 12 13
Insert DNA 1.2 1.2 --- 1.0 1.0 1.0 --- 2.1 3.7 --- 2.1 3.7 ---
Vector DNA 0.8 0.8 0.8 4.0 4.0 4.0 4.0 1.9 1.9 1.9 1.5 1.5 1.5
Ad BglII/EcoRI --- --- --- --- --- --- --- 0.4 0.4 0.4 0.4 0.4 0.4
Ad PstI/BglII --- --- --- --- --- --- --- 0.4 0.4 0.4 0.4 0.4 0.4
2x lgn buf (Roche) 5.0 5.0 5.0 6.0 6.0 6.0 6.0 7.4 7.4 7.4 7.0 7.0 7.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
dH2O 2.0 2.0 3.2 --- --- --- 1.0 1.6 --- 3.7 1.6 --- 3.7
  10μL 10μL 10μL 12μL 12μL 12μL 12μL 14.8μL 14.8μL 14.8μL 14μL 14μL 14μL

--> For reactions 8-13, add everything except the vector DNA, R.T./ 10 min.; then add vector, R.T. 10 min.
--> Add 20 μL Z-DH5α



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