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

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=Date=
=06/05/2013=


'''List title'''
'''Building a Reporter Gene to Determine Chromatin Protein Function (Ligation into the mammalian vector)'''
# List items
 
 
*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 KAH182-KAH201(N/S)/1901.
 
 
'''Digests''' <br>
 
 
[[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]]
 
{| {{table}} cellspacing="3"  width=350px
| Plasmid DNA || 15.00 μl
|-
| NotI  || 1.0 μl
|-
| SpeI || 1.0 μl
|-
| 10x FastDigest buffer + green loading dye || 3.00 μl
|-
| dH<sub>2</sub>O || 10.00 μl
|-
| &nbsp; || 30.0 μl total
|-
| Incubate at 37°C for 10 minutes.
|}
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
* '''Ligations'''
{| {{table}} cellspacing="3" <!-- Ligations table -->
|- bgcolor=#cfcfcf
| Ligation || <font color="blue"><u>
|-
| 1. E. coli BL21  KAH182-KAH201(N/S)/size, 21.00 ng + V0200(N/S)/size, 43.00ng || <font color="blue">KAH182/KAH201 3:1 Six Colonies</font>
|-
| 2. E. coli BL21  KAH182-KAH201(N/S)/size, 21.00 ng + V0200(N/S)/size, 43.00ng  || <font color="blue">KAH182/KAH201 4:1 One Colony</font>
|-
| 3. E. coli BL21  KAH182-KAH201(N/S)/size, 21.00 ng + V0200(N/S)/size, 43.00ng || <font color="blue">KAH182/KAH201 5:1 One Colony</font> 
|-
| 4. E. coli BL21  KAH182-KAH201(N/S)/size, 21.00 ng + V0200(N/S)/size, 43.00ng || <font color="blue">KAH182/KAH201 10:1 One Colony</font> 
|-
| 5. KAH201(S/P)/size, 36.29ng (Control Plate)|| <font color="blue">No Colony</font>
 
|}
 
* 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
 
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
| &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  || ---
|-
| 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
|-
| dH<sub>2</sub>O    || 1.16 || 0.75 || 0.34  ||  --- || 2.38
|-
| &nbsp;            || 10.00 μL || 10.00 μL || 10.00μl || 12.40μL || 10μL
|}
 
* The incubation time for the ligation process was 15min at room temperature.
* Fast transformation, 15 min on ice.

Revision as of 12:59, 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 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
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
  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.
  • Fast transformation, 15 min on ice.
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