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

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




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[[Image:KAH182-KAH201-V0200-S-N Digestion.jpg|thumb|350px| (1) KAH182-KAH201, 1901 bp, (2) V0200, 4442 bp, Both 1 and 2 are cut with Spe1 and Not11]]
[[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]]


{| {{table}} cellspacing="3"  width=350px  
{| {{table}} cellspacing="3"  width=350px  
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| Incubate at 37°C for 10 minutes.
| Incubate at 37°C for 10 minutes.
|}
|}




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{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
| &nbsp;            || 1 (3:1)    || 2 (4:1)  || 3 (5:1)  ||4 (10:1) || 5 (- Ctrl)
| Reactions &nbsp;            || 1 (3:1)    || 2 (4:1)  || 3 (5:1)  ||4 (10:1) || 5 (- Ctrl)
|-
|-
| Insert DNA        || 1.22 || 1.63 || 2.04  || 4.08  || ---
| Insert DNA        || 1.22 || 1.63 || 2.04  || 4.08  || ---
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* The incubation time for the ligation process was 15min at room temperature.
* The incubation time for the ligation process was 15min at room temperature.
* Fast transformation, 15 min on ice.
* 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.

Revision as of 13:10, 6 May 2013

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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
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.
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