User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/11/16

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(11/16/12)
Current revision (15:46, 26 November 2012) (view source)
(11/16/12)
 
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# KAH151/pT7CFE1-CHis: KAH191/(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
-
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>
-
> 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 -->
-
|-valign="top"
+
|- valign="top"
| <u>Reagent</u> || <u>Volume</u> || &nbsp;
| <u>Reagent</u> || <u>Volume</u> || &nbsp;
-
| rowspan="9" | <!--- [[Image:image.tif|350px|Cloning digest ]]<br>30 μL/lane, 1% agarose --->
+
| rowspan="9" |   [[Image:PcTF_in-vitro_Expression.png‎|350px|Cloning digest ]]<br>30 μL/lane, 1% agarose
|-
|-
| DNA (plasmid) || 25.0 μL
| DNA (plasmid) || 25.0 μL
Line 49: Line 55:
-
> Measure concentration(s)
+
'''Measure concentration(s)'''
{| class="wikitable" border="0" cellspacing="3" <!-- [DNA] table -->
{| class="wikitable" border="0" cellspacing="3" <!-- [DNA] table -->
|-
|-
| <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) || --- || 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)
-
> Ligations (trouble shooting suggestions)
+
* 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)
Line 66: Line 85:
| &nbsp;            || 1    || 2    || 3    || 4    || 5    || 6    || 7    || 8   
| &nbsp;            || 1    || 2    || 3    || 4    || 5    || 6    || 7    || 8   
|-
|-
-
| Insert DNA        || --- || --- || --- || --- || --- || --- || --- || ---
+
| Insert Name      || KAH109 || KAH111 || KAH112 || KAH115 || KAH148 || KAH150 || KAH151 || N/A
|-
|-
-
| Vector DNA        || --- || --- || --- || --- || --- || --- || --- || ---
+
| 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  
| 2x lgn buf (Roche) || 5.0  || 5.0  || 5.0  || 5.0  || 5.0  || 5.0  || 5.0  || 5.0  
Line 74: Line 95:
| 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
|}
|}
 +
 +
'''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

Current revision

PcTF Subcloning in E. coli Main project page
Previous entry      Next entry

11/16/12

  • ---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

  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   Cloning digest
30 μL/lane, 1% agarose
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) --- 1.9 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)
  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
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

Plates Nomenclature

  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, (KAH151)
  8. CTRL
  • 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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