User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/04/22

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Building a Reporter Gene to Determine Chromatin Protein Function (Ligation into the mammalian vector)


  • Ligation of construct KAH182-KAH201 and mammalian vector V0200 and cloning in DH5α-T.
  • KAH182 = BBa_S04739 and KAH201 = BBa_S04745 and V0200 = BBa_J176121.
  • Assembly strategy: The backbone V0200(N/S)/4442 , the insert is KAH182-KAH201(N/S)/1901.


Digests


(1) KAH182-KAH201, 1901 bp, (2) V0200, 4442 bp, Both 1 and 2 are cut with Spe1 and Not11
(1) KAH182-KAH201, 1901 bp, (2) V0200, 4442 bp, Both 1 and 2 are cut with Spe1 and Not11
Plasmid DNA 25.00 μl
NotI 1.0 μl
SpeI 1.0 μl
10x FastDigest buffer + green loading dye 3.00 μl
dH2O 0.00 μl
  30.0 μl total
Incubate at 37°C for 10 minutes.









  • Ligations
Ligation
1. E. coli DH5α-T KAH182-KAH201(N/S)/size, 9.00 ng + V0200(N/S)/size, 48.00ng KAH182/KAH201 3:1 No Colonies
2. E. coli DH5α-T KAH182-KAH201(N/S)/size, 9.00 ng + V0200(N/S)/size, 48.00ng KAH182/KAH201 4:1 One Colony
3. E. coli DH5α-T KAH182-KAH201(N/S)/size, 9.00 ng + V0200(N/S)/size, 48.00ng KAH182/KAH201 4:1 one Colony
4. KAH201(S/P)/size, 36.29ng (Control Plate) One Colonies and lots of satellite colonies in the middle of the pate
  • Calculations are for the ng of insert we need to get a 3:1, 4:1 and 5:1 ratios of insert molecules to 20 ng vector molecules
  1 (3:1) 2 (4:1) 3 (5:1) 4 (- Ctrl)
Insert DNA 2.85 3.80 4.76 ---
Vector DNA 1.00 1.00 1.00 1.00
2x lgn buf (Roche) 5.00 5.80 6.76 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0
dH2O 0.15 0.00 0.00 3.00
  10.00 μL 11.60 μL 13.52μl 10μL
  • The incubation time for the ligation process was 15min at room temperature.
  • Fast transformation, 15 min on ice.

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