User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/11/16: 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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''' | *'''[[User:Karmella Haynes|---Karmella]] 13:51, 16 November 2012 (EST)''': Here is my suggested procedure for trying the PcTF fusion cloning... | ||
# | |||
---- | |||
'''Assemblies''': Shown as Part/cuts/purified fragment size | |||
# KAH109/pT7CFE1-CHis: KAH109/(E/P)/1132 + pT7CFE1-CHis/(E/P)/3600 | |||
# KAH111/pT7CFE1-CHis: KAH184/(E/S)/1123 + " | |||
# KAH112/pT7CFE1-CHis: KAH187/(E/S)/1123 + " | |||
# KAH115/pT7CFE1-CHis: KAH188/(E/S)/931 + " | |||
# KAH148/pT7CFE1-CHis: KAH189/(E/S)/967 + " | |||
# KAH150/pT7CFE1-CHis: KAH190/(E/S)/967 + " | |||
# KAH151/pT7CFE1-CHis: KAH191/(E/S)/967 + " | |||
* Note: KAH109 is being re-done as an internal control to trouble-shoot the ligation<br> | |||
* Note: KAH111-KAH151 are all cut and purified already | |||
'''Digest (Fermentas FD)'''<br> | |||
# pT7CFE1-CHis, E/P | |||
# KAH109 E/P | |||
# KAH111 E/P | |||
# KAH112 E/P | |||
# KAH115 E/P | |||
# KAH148 E/P | |||
# KAH150 E/P | |||
# KAH151 E/P | |||
{| class="wikitable" border="0" cellspacing="3" <!-- Digest rxn. table --> | |||
|- valign="top" | |||
| <u>Reagent</u> || <u>Volume</u> || | |||
| rowspan="9" | [[Image:PcTF_in-vitro_Expression.png|350px|Cloning digest ]]<br>30 μL/lane, 1% agarose | |||
|- | |||
| DNA (plasmid) || 25.0 μL | |||
|- | |||
| 10x buffer || 3.0 | |||
|- | |||
| EcoRI || 1.0 | |||
|- | |||
| PstI || 1.0 | |||
|- | |||
| dH<sub>2</sub>O || 0 | |||
|- | |||
| || 30 μL --> 37°C/ ~30 min. | |||
|} | |||
'''Measure concentration(s)''' | |||
{| class="wikitable" border="0" cellspacing="3" <!-- [DNA] table --> | |||
|- | |||
| <u>Sample</u> || <u>OD260</u> || <u>260/280</u> || <u>ng/μL</u> | |||
|- | |||
| 1. pT7CFE1-CHis (E/P) || --- || 1.829 || 57.344 | |||
|- | |||
| 2. KAH109 (E/P) || --- || 2.354 || 36.072 | |||
|- | |||
| 3. KAH111 (E/P) || --- || 2.238 || 15.003 | |||
|- | |||
| 4. KAH112 (E/P) || --- || 1.862 || 22.857 | |||
|- | |||
| 5. KAH115 (E/P) || --- || 2.096 || 18.312 | |||
|- | |||
| 6. KAH148 (E/P) || --- || 2.389 || 23.735 | |||
|- | |||
| 7. KAH150 (E/P) || --- || 1.99 || 19.822 | |||
|- | |||
| 8. KAH151 (E/P) || --- || 1.9 || 19.701 | |||
|} | |||
'''Ligations''' (trouble shooting suggestions) | |||
* Note: Use 50 ng of vector this time (instead of 20) | |||
* Note: Use a 3:1 ratio of insert to vector... x μL insert = 3 * (bp of insert/ bp of vector) * 50 ng vector / (concentration of insert ng/μL) | |||
{| class="wikitable" border="0" cellspacing="3" <!-- Ligation rxn table --> | |||
| || 1 || 2 || 3 || 4 || 5 || 6 || 7 || 8 | |||
|- | |||
| Insert Name || KAH109 || KAH111 || KAH112 || KAH115 || KAH148 || KAH150 || KAH151 || N/A | |||
|- | |||
| Insert DNA || 1.3 || 3.12 || 2.05 || 2.12 || 1.7 || 2.03 || 2.05 || 0 | |||
|- | |||
| Vector DNA || 0.87 || 0.87 || 0.87 || 0.87 || 0.87 || 0.87 || 0.87 || 0.87 | |||
|- | |||
| 2x lgn buf (Roche) || 5.0 || 5.0 || 5.0 || 5.0 || 5.0 || 5.0 || 5.0 || 5.0 | |||
|- | |||
| T4 ligase (NEB) || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 | |||
|- | |||
| dH<sub>2</sub>O || 1.83 || 0.01 || 1.08 || 1.01 || 1.43 || 1.1 || 1.08 || 3.13 | |||
|- | |||
| || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL | |||
|} | |||
'''Plates Nomenclature | |||
# BD-116, (KAH109) | |||
# BD-117, (KAH111) | |||
# BD-118, (KAH112) | |||
# BD-119, (KAH115) | |||
# BD-120, (KAH148) | |||
# BD-121, (KAH150) | |||
# BD-122, (KAH151) | |||
# CTRL | |||
* Add total ligation reaction to 30 μL DH5α-Turbo cells | |||
* Follow routine "fast" transformation procedure | |||
* Plate onto 100 ug/mL Amp agar plates |
Latest revision as of 22:16, 26 September 2017
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11/16/12
Assemblies: Shown as Part/cuts/purified fragment size
Measure concentration(s)
Plates Nomenclature
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