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, The new construct is named: BD002.
*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''' <br>
 
 
[[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
| 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 BD001 (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  BD001 (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  BD001 (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  BD001 (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. V0200 (N/S)/size, 43.00ng (Negative 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 -->
| 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  || ---
|-
| 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.
* 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.
 
'''Confirm The Assembly'''
 
[[Image:Ligation Confirmation.jpg|thumb|350px| Two seperate bands in each column show the vector backbone and the insert, 4442 bps and the BD001 (KAH201+KAH182) (1901) constructed gene.Backbone, 1901 bps, Cut with SpeI and NotI. First column: Ladder,  columns 1-5 are 3:1 ratio colonies, column6 is 4:1 ratio, column 7 is 5:1 ratio and column 8 is 10:1]]
{| {{table}} cellspacing="3" width=400px
| Plasmid DNA || 2.0 μl
|-
| EcoR1  || 1.0 μl
|-
| Pst1 || 1.0 μl
|-
| 10x FastDigest buffer + green loading dye || 1.5 μl
|-
| dH<sub>2</sub>O || 9.5 μl
|-
| &nbsp; || 15.0 μl total
|-
| Incubate at 37°C for 10 minutes.
|}
 
# Make the (1%) agarose glee and add 15μl  of restricted vector in one well and 10 μl  of ladder.

Revision as of 13:42, 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, The new construct is named: BD002.
  • 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 BD001 (KAH182-KAH201) (N/S)/size, 21.00 ng + V0200(N/S)/size, 43.00ng KAH182/KAH201 3:1 Six Colonies
2. E. coli BL21 BD001 (KAH182-KAH201)(N/S)/size, 21.00 ng + V0200(N/S)/size, 43.00ng KAH182/KAH201 4:1 One Colony
3. E. coli BL21 BD001 (KAH182-KAH201) (N/S)/size, 21.00 ng + V0200(N/S)/size, 43.00ng KAH182/KAH201 5:1 One Colony
4. E. coli BL21 BD001 (KAH182-KAH201)(N/S)/size, 21.00 ng + V0200(N/S)/size, 43.00ng KAH182/KAH201 10:1 One Colony
5. V0200 (N/S)/size, 43.00ng (Negative 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.

Confirm The Assembly

Two seperate bands in each column show the vector backbone and the insert, 4442 bps and the BD001 (KAH201+KAH182) (1901) constructed gene.Backbone, 1901 bps, Cut with SpeI and NotI. First column: Ladder, columns 1-5 are 3:1 ratio colonies, column6 is 4:1 ratio, column 7 is 5:1 ratio and column 8 is 10:1
Plasmid DNA 2.0 μl
EcoR1 1.0 μl
Pst1 1.0 μl
10x FastDigest buffer + green loading dye 1.5 μl
dH2O 9.5 μl
  15.0 μl total
Incubate at 37°C for 10 minutes.
  1. Make the (1%) agarose glee and add 15μl of restricted vector in one well and 10 μl of ladder.
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