User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/11/20

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(11/20/12)
Current revision (15:01, 3 December 2012) (view source)
(11/20/12)
 
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|- valign="top"
|- valign="top"
| <u>Reagent</u> || <u>Volume</u> || &nbsp;
| <u>Reagent</u> || <u>Volume</u> || &nbsp;
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| rowspan="9" | <!--- [[Image:image.tif|350px|Cloning digest ]]<br>30 μL/lane, 1% agarose --->
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| rowspan="9" | [[Image:PcTF_in-vitro_Expression.png‎ |thumb|350px| 1. KAH109, 2. KAH111, 3. KAH112, 4. KAH115, 5. KAH148, 6. KAH150, 7. KAH151  8. Human Vector PT7CFE1-CHis(3600 bp) Cut with EcoRI and PstI]]
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<!--- [[Image:PcTF_in-vitro_Expression.png‎|350px|Cloning digest ]]<br>30 μL/lane, 1% agarose --->
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| DNA (plasmid) || 25.0 μL
| DNA (plasmid) || 25.0 μL
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| <u>Sample</u> || <u>OD260</u> || <u>260/280</u> || <u>ng/μL</u>
| <u>Sample</u> || <u>OD260</u> || <u>260/280</u> || <u>ng/μL</u>
|-
|-
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| 1. pT7CFE1-CHis (E/P) || 57.344 || 1.829 || ---
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| 1. pT7CFE1-CHis (E/P) || --- || 1.829 || 57.344
|-
|-
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| 2. KAH109, (E/P) || --- || 36.072 || 2.354
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| 2. KAH109, (E/P) || --- || 2.354 || 36.072
|-
|-
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| 3. KAH111, (E/P) || --- || 15.003 || 2.238
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| 3. KAH111, (E/P) || --- || 2.238 || 15.003
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|-
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| 4. KAH112, (E/P) || --- || 22.857 || 1.862
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| 4. KAH112, (E/P) || --- || 1.862 || 22.857
|-
|-
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| 5. KAH115, (E/P) || --- || 18.312 || 2.096
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| 5. KAH115, (E/P) || --- || 2.096 || 18.312
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|-
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| 6. KAH148, (E/P) || --- || 23.735 || 2.389
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| 6. KAH148, (E/P) || --- || 2.389 || 23.735
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|-
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| 7. KAH150, (E/P) || --- || 19.822 || 1.99
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| 7. KAH150, (E/P) || --- || 1.99 || 19.822
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|-
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| 8. KAH151, (E/P) || --- || 19.701 || 1.90
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| 8. KAH151, (E/P) || --- || 1.90 || 19.701
|}
|}

Current revision

PcTF Subcloning in E. coli Main project page
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11/20/12


Assemblies: Shown as Part/cuts/purified fragment size

  1. KAH109/pT7CFE1-CHis: KAH109/(E/P)/1132 + pT7CFE1-CHis/(E/P)/3600
  2. KAH111/pT7CFE1-CHis: KAH184/(E/S)/1123 + "
  3. KAH112/pT7CFE1-CHis: KAH187/(E/S)/1123 + "
  4. KAH115/pT7CFE1-CHis: KAH188/(E/S)/931 + "
  5. KAH148/pT7CFE1-CHis: KAH189/(E/S)/967 + "
  6. KAH150/pT7CFE1-CHis: KAH190/(E/S)/967 + "
  7. KAH151/pT7CFE1-CHis: KAH191/(E/S)/967 + "
  • Note: KAH109 is being re-done as an internal control to trouble-shoot the ligation
  • Note: KAH111-KAH151 are all cut and purified already


Digest (Fermentas FD)

  1. pT7CFE1-CHis, E/P
  2. KAH109, E/P
  3. KAH111, E/P
  4. KAH112, E/P
  5. KAH115, E/P
  6. KAH148, E/P
  7. KAH150, E/P
  8. KAH151, E/P
Reagent Volume  
1. KAH109, 2. KAH111, 3. KAH112, 4. KAH115, 5. KAH148, 6. KAH150, 7. KAH151  8. Human Vector PT7CFE1-CHis(3600 bp) Cut with EcoRI and PstI
1. KAH109, 2. KAH111, 3. KAH112, 4. KAH115, 5. KAH148, 6. KAH150, 7. KAH151 8. Human Vector PT7CFE1-CHis(3600 bp) Cut with EcoRI and PstI
DNA (plasmid) 25.0 μL
10x buffer 3.0
EcoRI 1.0
PstI 1.0
dH2O 0
  30 μL --> 37°C/ ~30 min.


Measure concentration(s)

Sample OD260 260/280 ng/μL
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) --- 1.90 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)
  1. BD-116 (KAH109)
  2. BD-117 (KAH111)
  3. BD-118 (KAH112)
  4. BD-119 (KAH115)
  5. BD-120 (KAH148)
  6. BD-121 (KAH150)
  7. BD-122 (KAH-151)
  8. CTRL
  1 2 3 4 5 6 7 8
Inser 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
dH2O 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


  • Add total ligation reaction to 30 μL DH5α-Turbo cells
  • Follow routine "fast" transformation procedure
  • Plate onto 100 ug/mL Amp agar plates
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