User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/11/01

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* '''Reverse Primer''': 5' <span style="color:#A9A9A9">gca</span> <span style="color:#FF0000">GCTCTTC</span> '''G''' GA<span style="color:#228B22">'''A'''</span>ACG + <span style="color:#800080">15 bps of bottom left strand</span>  
* '''Reverse Primer''': 5' <span style="color:#A9A9A9">gca</span> <span style="color:#FF0000">GCTCTTC</span> '''G''' GA<span style="color:#228B22">'''A'''</span>ACG + <span style="color:#800080">15 bps of bottom left strand</span>  
-
Forward Primer: 5’ gca GCTCTTC G TTC  25 pbs of top right
+
'''PCR Reactions'''
 +
{| class="wikitable" border=1 cellpadding="7" cellspacing="0"
 +
| align="center" style="background:#f0f0f0;"|'''Reagents'''
 +
| align="center" style="background:#f0f0f0;"|'''Volume'''
 +
| align="center" style="background:#f0f0f0;"|'''Reaction Conditions'''
 +
|-
 +
|{| DNA Template (ng)||200 || rowspan="6" |
 +
* 95°C, 3 min.
 +
* [95°C, 30 sec.; '''45'''°C, 40 sec.; 72°C, '''6''' min.] x35
 +
* 72°C, 3 min.
 +
* 4°C, ∞
 +
|-
 +
| Forward Promer (µL)||1
 +
|-
 +
| Reverse Primer (µL)||1
 +
|-
 +
| 2X GoTag (µL)||50
 +
|-
 +
| dH2O (µL)||22.8
 +
|-
 +
| Tolal (µL)||50
 +
|}
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Reverse Primer 5’ gca GCTCTTC G GAAACG 25 pbs of bottom left
 
*'''[[User:Behzad Damadzadeh|Behzad Damadzadeh]] 13:34, 4 November 2013 (EST)''':
*'''[[User:Behzad Damadzadeh|Behzad Damadzadeh]] 13:34, 4 November 2013 (EST)''':

Revision as of 15:44, 4 November 2013

PcTF Subcloning in E. coli Main project page
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PCR Based Mutagenesis Protocol

It is fast and efficient method using type IIs restriction enzymes. With designing a pair of mutagenesis primers that the mutated sequence is near and downstream of the recognition site of the type IIs restriction enzyme. PCR will linearize the plasmid with two blunt ends. Enzyme digestion will produce two sticky and complementary ends that will ligate together and generate the mutated plasmid. Here this method would be applied to mutate the BSMBI cut site within the hygromycine resistant gene of V0200 mammalian vector. Primers are designed for SapI restriction enzyme:

Here we plan to mutate the BSMBI cut site: CGTCTC to CGTTTC Design Primers:

  • Forward Primer: 5' gca GCTCTTC G TTC + 15 bps of top right strand
  • Reverse Primer: 5' gca GCTCTTC G GAAACG + 15 bps of bottom left strand

PCR Reactions

Reagents Volume Reaction Conditions
DNA Template (ng)200
  • 95°C, 3 min.
  • [95°C, 30 sec.; 45°C, 40 sec.; 72°C, 6 min.] x35
  • 72°C, 3 min.
  • 4°C, ∞
Forward Promer (µL)1
Reverse Primer (µL)1
2X GoTag (µL)50
dH2O (µL)22.8
Tolal (µL)50



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