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

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Design Primers:
Design Primers:
-
* '''Forward Primer''': 5' <span style="color:#A9A9A9">gca</span> <span style="color:#FF0000">GCTCTTC</span> '''G''' <span style="color:#228B22">'''T'''</span>TC + <span style="color:#800080">15 bps of top right strand</span>  
+
* '''Forward Primer''': 5' <span style="color:#A9A9A9">gca</span> <span style="color:#FF0000">GCTCTTC</span> '''G''' <span style="color:#228B22">'''T'''</span>TC + <span style="color:#800080">25 bps of top right 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>  
+
* '''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">25 bps of bottom left strand</span>  
'''PCR Reactions'''
'''PCR Reactions'''
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|{| DNA Template (ng)||200 || rowspan="6" |
|{| DNA Template (ng)||200 || rowspan="6" |
* 95°C, 3 min.
* 95°C, 3 min.
-
* [95°C, 30 sec.; '''45'''°C, 40 sec.; 72°C, '''6''' min.] x35
+
* [95°C, 30 sec.; '''45'''°C, 40 sec.; 72°C, * min (1 min/kb plasmid length)]] x35
* 72°C, 3 min.
* 72°C, 3 min.
* 4°C, ∞  
* 4°C, ∞  
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| Reverse Primer (µL)||1
| Reverse Primer (µL)||1
|-
|-
-
| 2X GoTag (µL)||50
+
| 2X GoTag (µL)||25
|-
|-
| dH2O (µL)||22.8
| dH2O (µL)||22.8
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|}
|}
 +
Run 5μL of each reaction on 1% agarose gel to confirm the plasmid linearization.
 +
DNA Purification Using the GenElute™ PCR Clean-Up Kit for rapid purification of double-stranded PCR amplification products. Measure the DNA concentration with plate reader. Cutting the SAPI sites to make the sticky ends for ligation:
 +
 +
'''SapI Digestion'''
 +
 +
{|class="wikitable" border=1 cellpadding="7" cellspacing="0"
 +
| align="center" style="background:#f0f0f0;"|'''Amplified DNA (500ng)'''
 +
| align="center" style="background:#f0f0f0;"|'''Volume'''
 +
|-
 +
| SapI μL||1.0
 +
|-
 +
| 10X Buffer μL ||5
 +
|-
 +
| dH2O μL||39.0
 +
|-
 +
|Total μL || 50
 +
|}
*'''[[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 16:20, 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 + 25 bps of top right strand
  • Reverse Primer: 5' gca GCTCTTC G GAAACG + 25 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, * min (1 min/kb plasmid length)]] x35
  • 72°C, 3 min.
  • 4°C, ∞
Forward Promer (µL)1
Reverse Primer (µL)1
2X GoTag (µL)25
dH2O (µL)22.8
Tolal (µL)50

Run 5μL of each reaction on 1% agarose gel to confirm the plasmid linearization.

DNA Purification Using the GenElute™ PCR Clean-Up Kit for rapid purification of double-stranded PCR amplification products. Measure the DNA concentration with plate reader. Cutting the SAPI sites to make the sticky ends for ligation:

SapI Digestion

Amplified DNA (500ng) Volume
SapI μL1.0
10X Buffer μL 5
dH2O μL39.0
Total μL 50


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