User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/04/18: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">PcTF Subcloning in E. coli</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">PcTF Subcloning in E. coli</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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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 | |||
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| Enzyme 1* || 1.0 μl | |||
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| Enzyme 2* || 1.0 μl | |||
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| 10x FastDigest buffer + green loading dye || 3.00 μl | |||
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| dH<sub>2</sub>O || 0.00 μl | |||
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| || 30.0 μl total | |||
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| Incubate at 37°C for 10 minutes. | |||
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* 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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* 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. | * 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 | |||
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| 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 | |||
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| 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. |
Latest revision as of 22:39, 26 September 2017
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04/18/2013Building a Reporter Gene to Determine Chromatin Protein Function
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