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

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'''Digest (Fermentas FD)'''<br>
'''Digest (Fermentas FD)'''<br>
# pT7CFE1-CHis, E/P
# 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 -->
{| class="wikitable" border="0" cellspacing="3" <!-- Digest rxn. table -->
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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>
|-
|-
| 1. pT7CFE1-CHis (E/P) || --- || --- || ---
| 1. pT7CFE1-CHis (E/P) || 57.344 || 1.829 || ---
|-
| 2. KAH109, (E/P) || --- || 36.072 || 2.354
|-
| 3. KAH111, (E/P) || --- || 15.003 || 2.238
|-
| 4. KAH112, (E/P) || --- || 22.857 || 1.862
|-
| 5. KAH115, (E/P) || --- || 18.312 || 2.096
|-
| 6. KAH148, (E/P) || --- || 23.735 || 2.389
|-
| 7. KAH150, (E/P) || --- || 19.822 || 1.99
|-
| 8. KAH151, (E/P) || --- || 19.701 || 1.90
|}
|}




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* Note: Use 50 ng of vector this time (instead of 20)
* 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)
* 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)
# BD-116 (KAH10
#
#
#
#
#
#
#


{| class="wikitable" border="0" cellspacing="3" <!-- Ligation rxn table -->
{| class="wikitable" border="0" cellspacing="3" <!-- Ligation rxn table -->
| &nbsp;            || 1    || 2    || 3    || 4    || 5    || 6    || 7    || 8   
| &nbsp;            || 1    || 2    || 3    || 4    || 5    || 6    || 7    || 8   
|-
|-
| Insert DNA        || --- || --- || --- || --- || --- || --- || --- || ---
|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        || --- || --- || --- || --- || --- || --- || --- || ---
| 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  
| 2x lgn buf (Roche) || 5.0  || 5.0  || 5.0  || 5.0  || 5.0  || 5.0  || 5.0  || 5.0  
Line 71: Line 104:
| T4 ligase (NEB)    || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  
| T4 ligase (NEB)    || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  
|-
|-
| dH<sub>2</sub>O    || --- || --- || --- || --- || --- || --- || --- || ---
| dH<sub>2</sub>O    || 1.83 || 0.01 || 1.08 || 1.01 || 1.43 || 1.1 || 1.08 || 3.13
|-
|-
| &nbsp;            || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL
| &nbsp;            || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL

Revision as of 11:58, 26 November 2012

PcTF Subcloning in E. coli <html><img src="/images/9/94/Report.png" border="0" /></html> 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  
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) 57.344 1.829 ---
2. KAH109, (E/P) --- 36.072 2.354
3. KAH111, (E/P) --- 15.003 2.238
4. KAH112, (E/P) --- 22.857 1.862
5. KAH115, (E/P) --- 18.312 2.096
6. KAH148, (E/P) --- 23.735 2.389
7. KAH150, (E/P) --- 19.822 1.99
8. KAH151, (E/P) --- 19.701 1.90



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 (KAH10
  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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