User:Karmella Haynes/Notebook/BioBrick cloning/2011/06/29

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06/29/11

  • ✓ Biobrick PCR: SP1A and SP1B (from s.d. mutated SP1AB)
  • ✓ Assemblies: KAH199 (spacer + TK), KAH200
  • ✓ SD mutagenesis: re-do mutagenesis of PstI site #2 in p65
  • ✓ Cultures/ minipreps: KAH187-198/MV8 (2); concentration of stock is low



Biobrick PCR: SP1A, SP1B
> Note: Last step, complete SP1AB mutation, was confirmed on 3/25/11

  1. KAH018: (1) SP1A/(X/P PCR)/540 + V0120/(X/P)/~3200 ✓
  2. KAH019: (2) SP1B/(X/P PCR)/840 + "

> PCR
--> Template: SP1AB Biobrick
--> Primers 1: BB_SP1A f/ BB_SP1A r
--> Primers 2: BB_SP1B f/ BB_SP1B r


Reagent Volume
DNA (plasmid) 1.0
10 μM primers 1.0
2x GoTaq 25.0
dH2O 23.0

--> BioRad DNA Engine, block A

  • 95°C/ 3 min.
  • [95°C/ 30 sec; 55°C/ 30 sec; 72 °C/ 1 min.] x30
  • 72 °C/ 3 min.
  • 4 °C/ ∞

> Zymo clean PCR; elute w/ 25 μL dH2O

> Digests (Fermentas FD)

Reagent Volume   PCR digest 06/29/11
30 μL/lane, 1% agarose
Expected: 2. SP1A = 540, 3. SP1B = 840
PCR DNA 25.0
10x buffer 3.0
XbaI 1.0
PstI 1.0
dH2O ---
  30 μL --> 37°C/ ~30 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. SP1A (X/P PCR) 0.912 1.86 45.6
2. SP1B (X/P PCR) 0.614 1.80 30.7


> Ligations

Ligation Plate results (lig : neg crtl) 06/30/11
1. SP1A (X/P)/540, 8 ng + V0120(X/P)/~3200, 25 ng SP1A (KAH018) >10:1 (Pick 2)
2. SP1B (X/P)/840, 13 ng + " SP1B (KAH019) >10:1 (Pick 2)
3. V0120(X/P)/ 25 ng  
  1 2 3
Insert DNA 0.5 0.5 ---
Vector DNA 1.0 1.0 1.0
2x lgn buf (Roche) 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0
dH2O 3.0 3.0 3.5
  10 μL 10 μL 10 μL

--> R.T./ ~15 min.; Add to 30 μL DH5α turbo



Assemblies
--> Note: DPRE has PstI sites

  1. KAH199: (1) Spacer B433/(E/S dp)/65 + (2) HSVtk TATA DDB81/(E/X)/56
  2. KAH200: DPRE KAH014/(E/S)/1740 ✓ + 5xGal4 KMC01/(E/X)/93 ✓

> Digests (Fermentas FD)
--> Specific notes

Reagent Volume   Hover name
30 μL/lane, 1% agarose; Lane 1 = DDB81(E/X)
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.


> Zymo clean
--> Spacer B433 (E/S) insert
--> Elute w/ 25 μL dH2O


> Measure conc.'s

Sample OD260 260/280 ng/μL
2. HSVtk TATA DDB81 (E/X) 1.677 1.87 83.9

--> Dilute 1:2 to get ~42 μg/μL


> Dephosphorylation (Roche)
--> Phosphatase-treat the Spacer (E/S) insert

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
--> Use max insert volume for ligation #1 (insert includes old vector)

Ligation Plate results (lig : neg crtl) 06/30/11
1. Spacer B433(E/S dp)/65, 1 ng + HSVtk TATA DB81(E/X)/~3256, 25 ng KAH199 >10:1 (Pick 2)
2. HSVtk TATA DB81(E/X)/ 25 ng  
3. DPRE KAH014(E/S)/1740, 26 ng + 5xGal4 KMC01(E/X)/~3293, 25 ng KAH200 1:1 (Pick 2)
4. 5xGal4 KMC01(E/X)/ 25 ng  
  1 2 3 4
Insert DNA 3.5 --- 1.0 ---
Vector DNA 0.5 0.5 0.5 0.5
2x lgn buf (Roche) 5.0 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0
dH2O --- 3.5 2.5 3.5
  10 μL 10 μL 10 μL 10 μL

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



Site-directed Mutagenesis
> Stratagene Quick Change mutagenesis kit.

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

--> Primer mut_p65 1

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/ ∞


(6/30/11)
> DpnI Digest (gets rid of methylated template DNA)

  1. p65 mutation
  2. 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


07/01/11
--> Results

  • p65: 2 colonies (1 on neg ctrl.)



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