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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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= | =04/18/2013= | ||
''' | '''Building a Reporter Gene to Determine Chromatin Protein Function''' | ||
# | |||
*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. | |||
'''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''' | |||
{| {{table}} cellspacing="3" <!-- Ligations table --> | |||
|- bgcolor=#cfcfcf | |||
| Ligation || <font color="blue"><u></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 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> | |||
|- | |||
| 4. KAH201(S/P)/size, 36.29ng (Control Plate)|| <font color="blue">No Colonies</font> | |||
|} | |||
* Calculations are for the ng of insert we need to get a 2:1, 3:1 and 4:1 ratios of insert molecules to 50 ng vector molecules | |||
{| {{table}} cellspacing="3" <!-- Ligation rxn table --> | |||
| || 1 (2:1) || 2 (3:1) || 3 (4:1) || 4 (- Ctrl) | |||
|- | |||
| Insert DNA || 3.11 || 4.66 || 6.21 || --- | |||
|- | |||
| Vector DNA || 1.37 || 1.37 || 1.37 || 1.37 | |||
|- | |||
| 2x lgn buf (Roche) || 5.48 || 7.03 || 8.58 || 5.0 | |||
|- | |||
| T4 ligase (NEB) || 1.0 || 1.0 || 1.0 || 1.0 | |||
|- | |||
| dH<sub>2</sub>O || 0.00 || 0.00 || 0.00 || 2.63 | |||
|- | |||
| || 10.96 μL || 14.06 μL || 17.16μl || 10μL | |||
|} | |||
* The incubation time for the ligation process was 15min at room temperature. | |||
* 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. |
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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