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

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07/07/11

  • ✓ Minipreps: mCherry, 5xGal4 retransformations
  • ✓ Assemblies: KAH204, 205, 206, 207, 208, 209
  • ✓ Site directed mutagenesis: p65 (new primers)
  • ✓ Digest: investigate weird miR-luc sensor constructs; check KAH196, 177, 198/MV8



Minipreps
> Check with E/P digests

Reagent Volume Expected:
1. mCherry = 705
2. 5xGal4 = 93
File:KAH 070711 gel1.tif
15 μL/lane; 1% agarose
DNA(plasmid) 3.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 8.5
  15 μL --> 37°C/ ~15 min.

Assemblies

  1. KAH204: (1) mCh/(S/P)/705 + (2) SP1AB/(X/P)/1617
  2. KAH205: " + (3) SP1A/(X/P)/540
  3. KAH206: " + (4) SP1B/(X/P)/840
  4. KAH207: (5) KAH203/(S/P)/155 + (8) KAH182/(X/P)/1675
  5. KAH208: (6) KAH201/(S/P)/203 + "
  6. KAH209: (7) KAH202/(S/P)/251 + "

> Digests (Fermentas FD)

Reagent Volume   File:KAH 070711 gel2.tif
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. mCh (S/P) 0.972 1.86 48.6
2. SP1AB (X/P) 0.170 6.31 8.5
3. SP1A (X/P) 0.268 2.50 13.4
4. SP1B (X/P) 0.298 2.46 14.9
5. KAH203 (S/P) 0.153 1.89 7.7
6. KAH201 (S/P) 0.128 2.53 6.4
7. KAH202 (S/P) 0.118 7.22 5.9
8. KAH182 (X/P) 0.120 3.76 6.0


> Ligations

Ligation Plate results (lig : neg crtl) 07/08/11
1. SP1AB(X/P)/1617, 17 ng + mCh(S/P)/~3905, 20 ng KAH204 >10:1 (Pick 2)
2. SP1A(X/P)/540, 6 ng + " KAH205 >10:1 (Pick 2)
3. SP1B(X/P)/840, 9 ng + " KAH206 >10:1 (Pick 2)
4. mCh(S/P)/ 20 ng  
5. KAH182(X/P)/1675, 20 ng + KAH203(S/P)/~3355, 20 ng KAH207 >10:1 (Pick 2)
6. KAH203(S/P)/ 20 ng  
7. KAH182(X/P)/1675, 20 ng + KAH201(S/P)/~3403, 20 ng KAH208 >10:1 (Pick 2)
8. KAH201(S/P)/ 20 ng  
9. KAH182(X/P)/1675, 19 ng + KAH202(S/P)/~3451, 20 ng KAH209 >10:1 (Pick 2)
10. KAH201(S/P)/ 20 ng  
  1 2 3 4 5 6 7 8 9 10
Insert DNA 2.5 0.5 1.0 --- 3.0 --- 3.0 --- 3.0 ---
Vector DNA 0.5 0.5 0.5 0.5 2.5 2.5 3.0 3.0 3.0 3.0
2x lgn buf (Roche) 5.0 5.0 5.0 5.0 6.5 6.5 7.0 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
dH2O 1.0 3.0 2.5 3.5 --- 3.0 --- 3.0 --- 3.0
  10 μL 10 μL 10 μL 10 μL 13 μL 13 μL 14 μL 14 μL 14 μL 14 μL



Site-directed Mutagenesis
> Stratagene Quick Change mutagenesis kit.

  1. p65 (~4 kb): tried to mutate w.t. clone (two PstI sites within single primer); last attempt mutated only the first site (3/25/11)

--> Try to mutate second PstI site with forward and reverse primers...

  1. Primer mut_p65 2 (plus strand)
  2. Primer mut_p65 2 (minus strand)
Reagent p65 w.t.
DNA (plasmid, ~100 ng) 1.0
10x buffer 2.5
Quick Solution 0.5
10 μM primer 1 1.0
dNTP mix 1.0
Quick Change enzyme mix 1.0
dH2O 18.0
  25 μL

--> BioRad PCR (Block A)

  • 95°C/ 1 min.
  • [95°C/ 1 min., 55°C/ 1 min., 65°C/ 8 min (2 min./kb)] x30
  • 65°C/ 1 min.
  • 4°C/ ∞


(7/08/11)
> DpnI Digest (gets rid of methylated template DNA)

  1. p65 + strand mutation
  2. p65 - strand mutation
  3. p65 control (DNA, 10x buffer, Quick soln. in 25 μL; no PCR)

--> Add 1 μL DpnI enzyme to each sample
--> 37°C/ 1 hr.
--> Transform 30 μL z-DH5α, use 10 μL each sample; Amp plates


07/09/11
--> Results

  • p65 +strand mut: 0 colonies (0 on neg ctrl.)
  • p65 -strand mut: 0 colonies (0 on neg ctrl.)



Minipreps
> Check with ApaLI
> Refer to 6/04/11 for correct 196, 198 bands (E/ApaLI)

Reagent Volume Expected:
MV8 bands = 1243, 1135, 288
1. KAH196/MV8 (6/05/11) = **2772, *1668, 711
2. KAH177/MV8 (1/020/11) = **2772, *1835, 711
3. KAH198/MV8 (6/05/11) = **2772, *1915, 711
*first frag. + 234
**last frag + 1655
File:KAH 070711 gel3.tif
15 μL/lane; 1% agarose
DNA(plasmid) 3.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 8.5
  15 μL --> 37°C/ ~15 min.

--> Everything looks correct. Use these minipreps for re-cloning final miR-luc sensor set.
--> Note: pre-cut vector KAH177/MV8 worked for 183-177/MV8 (see 6/07/11); continue using this vector)
--> Re-cut 196 and 198/MV8, throw out old cut vectors