User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/04/18: Difference between revisions
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'''Building a Reporter Gene to Determine Chromatin Protein Function''' | '''Building a Reporter Gene to Determine Chromatin Protein Function''' | ||
*Ligation of KAH182 = BBa_S04739 and KAH201 = BBa_S04745 and cloning in DH5α-T. | *Ligation of KAH182 = BBa_S04739 and KAH201 = BBa_S04745 and cloning in DH5α-T. | ||
*Assembly strategy: The backbone KAH201(S/P)/3403 , the insert is KAH182(X/P)/1675. | *Assembly strategy: The backbone KAH201(S/P)/3403 , the insert is KAH182(X/P)/1675. | ||
'''Digests''' <br> | |||
[[Image:KAH201-KAH182 Ligation.jpg|thumb|400px| (1,2) KAH182, 1675 bp, Cut with Xba1 and Pst1, (3,4) KAH201, 3403 bp, Cut with Spe1 and Pst1]] | |||
{| {{table}} cellspacing="3" width=350px | |||
| Plasmid DNA || 25.00 μl | |||
|- | |||
| Enzyme 1* || 1.0 μl | |||
|- | |||
| Enzyme 2* || 1.0 μl | |||
|- | |||
| 10x FastDigest buffer + green loading dye || 3.00 μl | |||
|- | |||
| dH<sub>2</sub>O || 0.00 μl | |||
|- | |||
| || 30.0 μl total | |||
|- | |||
| Incubate at 37°C for 10 minutes. | |||
|} | |||
* Enzyme 1 for KAH182 is Xba1 and for KAH201 is Spe1. | |||
* Enzyme 2 for KAH182 and KAH201 is Pst1. | |||
# Make the (1%) agarose glee and add 30μl of restricted vector in one well and 10 μl of ladder. | |||
* Ligations | * '''Ligations''' | ||
{| {{table}} cellspacing="3" <!-- Ligations table --> | {| {{table}} cellspacing="3" <!-- Ligations table --> | ||
|- bgcolor=#cfcfcf | |- bgcolor=#cfcfcf | ||
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| 1. E. coli DH5α-T KAH182(X/P)/size, 15.85 ng + KAH201(S/P)/size, 36.29ng || <font color="blue">KAH182/KAH201 2:1 No Colonies</font> | | 1. E. coli DH5α-T KAH182(X/P)/size, 15.85 ng + KAH201(S/P)/size, 36.29ng || <font color="blue">KAH182/KAH201 2:1 No Colonies</font> | ||
|- | |- | ||
| 2. E. coli DH5α-T KAH182(X/P)/size, 15.85 ng + KAH201(S/P)/size, 36.29ng || <font color="blue">KAH182/KAH201 3:1 | | 2. E. coli DH5α-T KAH182(X/P)/size, 15.85 ng + KAH201(S/P)/size, 36.29ng || <font color="blue">KAH182/KAH201 3:1 One Colony</font> | ||
|- | |- | ||
| 3. E. coli DH5α-T KAH182(X/P)/size, 15.85 ng + KAH201(S/P)/size, 36.29ng || <font color="blue">KAH182/KAH201 4:1 Two Colonies</font> | | 3. E. coli DH5α-T KAH182(X/P)/size, 15.85 ng + KAH201(S/P)/size, 36.29ng || <font color="blue">KAH182/KAH201 4:1 Two Colonies</font> | ||
|- | |- | ||
| 4. KAH201(S/P)/size, 36.29ng (Control Plate)|| | | 4. KAH201(S/P)/size, 36.29ng (Control Plate)|| <font color="blue">No Colonies</font> | ||
|} | |} | ||
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| T4 ligase (NEB) || 1.0 || 1.0 || 1.0 || 1.0 | | T4 ligase (NEB) || 1.0 || 1.0 || 1.0 || 1.0 | ||
|- | |- | ||
| dH<sub>2</sub>O || 0.00 || 0.00 || 0.00 || | | dH<sub>2</sub>O || 0.00 || 0.00 || 0.00 || 2.63 | ||
|- | |- | ||
| || 10.96 μL || 14.06 μL || 17.16μl || 10μL | | || 10.96 μL || 14.06 μL || 17.16μl || 10μL | ||
|} | |} | ||
* The incubation time for the ligation process was | * The incubation time for the ligation process was 15min at room temperature. | ||
* Fast transformation, | * Fast transformation, 15 min on ice. | ||
'''Confirm The Assembly''' | |||
[[Image:KAH201-KAH182-Ligation Confirmation.png|thumb|350px| Two seperate bands in each column show the vector backbone and the insert, 4400 bps and the KAH201+KAH182 (226+1675) constructed gene.Backbone, 1901 bps, Cut with EcoRI and PstI. First column: Ladder, Second collumn: 3:1 ratio, Third and fourth column: 4:1 ratio (colony A and B)]] | |||
{| {{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 | |||
|- | |||
| || 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 13:22, 6 May 2013
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04/18/2013Building a Reporter Gene to Determine Chromatin Protein Function
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