User:Karmella Haynes/Notebook/BioBrick cloning/2010/01/28

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01/28/10

  • ✓ Assembly: KAH139/V0201
  • ✓ PCR SOEing: EF1α promoter (mutate two PstI sites)

PCR SOEing
> EF-1α promoter (for miR sensor optimization); has 2 PstI sites
> Step 1: mutate one of the PstI sites (later, use the mutated BioBrick for mutation of other PstI)

  1. Fwd EF1A / EF-1a Pst1 r, amplicon 1 (1st PstI site)
  2. EF-1a Pst1 f / Rev EF1A, amplicon 2 (1st PstI site)
  3. Fwd EF1A / EF-1a Pst2 r, amplicon 1b (2nd PstI site)*
  4. EF-1a Pst2 f / Rev EF1A, amplicon 2b (2nd PstI site)*

--> *Done in case PCR for mutating 1st PstI site fails

> PCR round 1

Reagent Volume Expected:
1. amplicon 1 = 337
2. amplicon 2 = 879
3. amplicon 1b = 861
4. amplicon 2b = 374
File:KAH 012810 gel1.tif
10 μL/lane; 1% agarose
DNA(plasmid) 0.5 (from Bruno)
10 μM primer mix 1.0
2x GoTaq green 25.0
dH2O 23.5
  50 μL

--> BioRad Block A

  • 95°C/ 3 min.
  • [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec.] x35
  • 72°C/ 3 min.
  • 4°C/ ∞

--> Clean bands 1 and 4 using Zymo clean & concentrator; elute w/ 20 μL dH2O

> Alternative Step 2
--> 1/2 fragment PCR didn't work, but mutation PCR for each end worked. Try using these as megaprimers (with EF-1α template) to create both PstI mutations at the same time.
--> Small population of amplicons should contain both mutations. Digestion w/ PstI should cut all products except for double-mutant fragments.

Reagent Volume Expected:
1184 bp
File:KAH 012810 gel2.tif
10 μL/lane; 1% agarose
DNA(plasmid) 0.5 (from Bruno)
amplicon 1 (clean) 5.0
amplicon 2b (clean) 5.0
2x GoTaq green 25.0
dH2O 14.5
  50 μL

--> BioRad Block A

  • 95°C/ 3 min.
  • [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec.] x35
  • 72°C/ 3 min.
  • 4°C/ ∞

--> Very faint product appears to be correct size. Repeat this PCR with 4 rxn's and 2.0 μL each megaprimer (tomorrow)



Assemblies

  1. KAH139/V0201: ✓ KAH139/(N/S)/2945 + ✓ V0201/(N/S)/5157

> Ligations

Ligation Plate results (lig : neg crtl) 01/29/10
1. KAH139(N/S)/2945, 29 ng + V0201(N/S, d.p.)/5157, 25 ng KAH139/V0201 10:1 (Pick 4)
2. V0201(N/S)/ 25 ng  
  1 2
Insert DNA 3.0 ---
Vector DNA 1.0 1.0
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O --- 3.0
  10μL 10μL --> R.T./ ~30 min.; Add 30 μL z-DH5α