User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/05/06

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(06/05/2013)
(06/05/2013)
Line 20: Line 20:
-
[[Image:Vector Insert Restriction Digestion.jpg|thumb|450px| (1 & 2) BD001 (KAH182-KAH201), 1901 bp, (3 & 4) V0200, 4442 bp, 1, 2, 3 and 4 are cut with Spe1 and Not11]]
+
[[Image:Vector Insert Restriction Digestion.jpg|thumb|400px| (1 & 2) BD001 (KAH182-KAH201), 1901 bp, (3 & 4) V0200, 4442 bp, 1, 2, 3 and 4 are cut with Spe1 and Not11]]
{| {{table}} cellspacing="3"  width=350px  
{| {{table}} cellspacing="3"  width=350px  

Revision as of 15:10, 6 May 2013

PcTF Subcloning in E. coli Main project page
Previous entry      Next entry

06/05/2013

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


  • Ligation of construct BD001(KAH182-KAH201) and mammalian vector V0200 and cloning in Bl21-DE3.
  • KAH182 = BBa_S04739 and KAH201 = BBa_S04745 and V0200 = BBa_J176121.
  • Assembly strategy: The backbone V0200(N/S)/4442 , the insert is BD001 (KAH182-KAH201) (N/S)/1901.


Digests


(1 & 2) BD001 (KAH182-KAH201), 1901 bp, (3 & 4) V0200, 4442 bp, 1, 2, 3 and 4 are cut with Spe1 and Not11
(1 & 2) BD001 (KAH182-KAH201), 1901 bp, (3 & 4) V0200, 4442 bp, 1, 2, 3 and 4 are cut with Spe1 and Not11
Plasmid DNA 15.00 μl
NotI 1.0 μl
SpeI 1.0 μl
10x FastDigest buffer + green loading dye 3.00 μl
dH2O 10.00 μl
  30.0 μl total
Incubate at 37°C for 10 minutes.







  • Ligations
Ligation
1. E. coli BL21 KAH182-KAH201(N/S)/size, 21.00 ng + V0200(N/S)/size, 43.00ng KAH182/KAH201 3:1 Six Colonies
2. E. coli BL21 KAH182-KAH201(N/S)/size, 21.00 ng + V0200(N/S)/size, 43.00ng KAH182/KAH201 4:1 One Colony
3. E. coli BL21 KAH182-KAH201(N/S)/size, 21.00 ng + V0200(N/S)/size, 43.00ng KAH182/KAH201 5:1 One Colony
4. E. coli BL21 KAH182-KAH201(N/S)/size, 21.00 ng + V0200(N/S)/size, 43.00ng KAH182/KAH201 10:1 One Colony
5. KAH201(S/P)/size, 36.29ng (Control Plate) No Colony
  • Calculations are for the ng of insert we need to get a 3:1, 4:1, 5:1 and 10:1 ratios of insert molecules to 20 ng vector molecules
Reactions   1 (3:1) 2 (4:1) 3 (5:1) 4 (10:1) 5 (- Ctrl)
Insert DNA 1.22 1.63 2.04 4.08 ---
Vector DNA 1.62 1.62 1.62 1.62 1.62
2x lgn buf (Roche) 5.00 5.00 5.00 5.70 5.00
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0
dH2O 1.16 0.75 0.34 --- 2.38
  10.00 μL 10.00 μL 10.00μl 12.40μL 10μL
  • The incubation time for the ligation process was 15min at room temperature.
  • Reaction number 1, 2, 3, 4, 5 40μL BL21 in 2.0 mL tubes; ice 2 min.; 42°C 45 sec.; add 800 μL SOC medium; shake @ 37°C 60 min.; pellet @ top speed 3 min.; resuspend in 100 μL amp liq. medium; plate on amp agar.

Personal tools