Team:SUSTC-Shenzhen/Notebook/CRISPR/UAS-Fill-in-second-time

From 2014.igem.org

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       name=UAS Fill-in (The second time)|
       name=UAS Fill-in (The second time)|
       date=2014/8/9|
       date=2014/8/9|
-
       goal=The same as the first Fill-in experiment}}
+
       goal=}}
-
<span lang="EN-US">Materials</span>
+
==Materials==
-
<span lang="EN-US">The same as the first Fill-in experiment</span>
+
The same as the first Fill-in experiment.
-
'''<span lang="EN-US">Methods:</span>'''
+
==Methods==
-
'''<span lang="EN-US">1. Restriction digestion for 5*UAS plasmid with EcoRI</span>'''（<span lang="EN-US">unit: ul</span>）
+
===Restriction digestion for 5*UAS plasmid with EcoRI (unit: ul)===
-
{|
+
-
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US"> </span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">Total  volume</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">10X  Buffer</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">DNA</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">EcoRI</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">ddH2O</span> 
+
-
+
-
|-
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">Poly A(168.8ng/ul)</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">20</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">2</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">2.5</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">1</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">14.5</span> 
+
-
+
-
|-
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">G-PB5(293.6ng/ul)</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">20</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">2</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">2</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">1</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">15</span> 
+
-
+
-
|}
+
-
<span lang="EN-US">Time: 2014.8.8 10:00pm ~ 2014.8.9 9:30am (
+
-
Incubate overnight)</span>
+
-
'''<span lang="EN-US">2. Electrophoresis to verify
+
{|class="table"
-
if most of the digested plasmid has been linearized</span>'''
+
!
-
 
+
!Total  volume
-
'''<span lang="EN-US">Loading system</span>：'''(<span lang="EN-US">unit: ul</span>)
+
!10X Buffer
-
{|
+
!DNA
-
   
+
!EcoRI
-
  |
+
!ddH2O
-
<nowiki>  </nowiki><span lang="EN-US">Total  volume/well</span>
+
|-
-
 
+
|Poly A(168.8ng/ul)
-
  |
+
|20
-
<nowiki>  </nowiki><span lang="EN-US">DNA</span>
+
|2
-
 
+
|2.5
-
  |
+
|1
-
<nowiki>  </nowiki><span lang="EN-US">Dye</span>
+
|14.5
-
 
+
|-
-
  |
+
|G-PB5(293.6ng/ul)
-
<nowiki>  </nowiki><span lang="EN-US">TAE</span> 
+
|20
-
+
|2
-
|-
+
|2
-
  |
+
|1
-
<nowiki>  </nowiki><span lang="EN-US">12</span>
+
|15
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">0.5</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">2</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">9.5</span> 
+
-
+
|}
|}
-
<span lang="EN-US">Running conditions: 130V,   30min</span>
 
-
'''<span lang="EN-US">Results of electrophoresis:</span>'''
+
Time: 2014.8.8 10:00pm ~ 2014.8.9 9:30am (Incubate overnight)
-
<span lang="EN-US"> </span>
+
===Electrophoresis to verify if most of the digested plasmid has been linearized===
-
<span lang="EN-US"> </span>
+
'''Loading system：'''(unit: ul)
 +
{|class="table"
 +
|Total  volume/well
 +
|DNA
 +
|Dye
 +
|TAE 
 +
|-
 +
|12
 +
|0.5
 +
|2
 +
|9.5
 +
|}
 +
Running conditions: 130V,   30min
-
<span lang="EN-US"> </span>
+
'''Results of electrophoresis:'''
-
<span lang="EN-US"> </span>
+
{{SUSTC-Image|wiki/images/6/63/SUSTC-Shenzhen-UAS-2nd-1.PNG}}
-
<span lang="EN-US"> </span>
+
From the picture, we can see that,
-
 
+
-
<span lang="EN-US"> </span>
+
-
 
+
-
<span lang="EN-US"> </span>
+
-
 
+
-
<span lang="EN-US"> </span>
+
-
 
+
-
<span lang="EN-US"> </span>
+
-
 
+
-
<span lang="EN-US"> </span>
+
-
 
+
-
<span lang="EN-US"> </span>
+
-
 
+
-
<span lang="EN-US">From the picture, we can see that,
+
the position of the band of digested plasmid is apparently lying behind that of
the position of the band of digested plasmid is apparently lying behind that of
the original plasmid which hasn’t been digested by EcoRI, indicating that most
the original plasmid which hasn’t been digested by EcoRI, indicating that most
-
of the plasmid in our digestion system has become linearized. </span>
+
of the plasmid in our digestion system has become linearized.  
-
'''<span lang="EN-US">3. Heat-inactivation for the
+
===Heat-inactivation for the digestion system & Cold shock to prevent self-ligation===
-
digestion system & Cold shock to prevent self-ligation</span>'''
+
-
<span lang="EN-US">1. Heat-inactivation 75</span>°<span lang="EN-US">C</span>，<span lang="EN-US">10min</span>
+
1. Heat-inactivation 75°C，10min
-
<span lang="EN-US">2. Chill on ice, 15min</span>
+
2. Chill on ice, 15min
-
'''<span lang="EN-US">4. Do fill-in with T4 DNA
+
===Do fill-in with T4 DNA polymerase===
-
polymerase</span>'''
+
-
'''<span lang="EN-US">1.
+
'''1. Discard 0.5ul DNA from the former inactivated digestion system, forming the
-
Discard 0.5ul DNA from the former inactivated digestion system, forming the
+
final 19ul system.'''
-
final 19ul system.</span>'''
+
-
<span lang="EN-US">Till now, the concentration of DNA for:</span>
+
Till now, the concentration of DNA for:
-
<span lang="EN-US">Poly A: 21.1ng/ul</span>
+
Poly A: 21.1ng/ul
-
<span lang="EN-US">            G-PB5: 29.36ng/ul</span>
+
G-PB5: 29.36ng/ul
-
'''<span lang="EN-US">2. Fill-insystem</span>：'''（<span lang="EN-US">unit: ul</span>）
+
'''2. Fill-insystem：(unit: ul)'''
-
{|
+
{|class="table"
-
+
|
-
  |
+
|Total  volume
-
<nowiki>  </nowiki><span lang="EN-US"> </span>
+
|DNA
-
 
+
|T4 DNA polymerase(3U/ul)
-
  |
+
|dNTP (10mM)
-
<nowiki>  </nowiki><span lang="EN-US">Total  volume</span>
+
|ddH2O
-
 
+
|-
-
  |
+
|Poly A(21.1ng/ul)
-
<nowiki>  </nowiki><span lang="EN-US">DNA</span>
+
|20
-
 
+
|19
-
  |
+
|0.2 (theoretical volume:0.13ul)
-
<nowiki>  </nowiki><span lang="EN-US">T4
+
|0.2
-
  DNA polymerase(3U/ul)</span>
+
|0.6
-
 
+
|-
-
  |
+
|G-PB5(29.36ng/ul)
-
<nowiki>  </nowiki><span lang="EN-US">dNTP (10mM)</span>
+
|20
-
 
+
|19
-
  |
+
|0.2 (theoretical volume:0.186ul)
-
<nowiki>  </nowiki><span lang="EN-US">ddH2O</span> 
+
|0.2
-
+
|0.6
-
|-
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">Poly
+
-
  A(21.1ng/ul)</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">20</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">19</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">0.2</span>
+
-
 
+
-
<span lang="EN-US">(theoretical volume:0.13ul)</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">0.2</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">0.6</span> 
+
-
+
-
|-
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">G-PB5(29.36ng/ul)</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">20</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">19</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">0.2</span>
+
-
 
+
-
<span lang="EN-US">(theoretical
+
-
  volume:0.186ul)</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">0.2</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">0.6</span> 
+
-
+
|}
|}
-
<span lang="EN-US">[According to the manual provided by NEB</span>：
 
-
<span lang="EN-US">The NEBuffer 2.1 for EcoRI is also suitable
+
[According to the manual provided by NEB：The NEBuffer 2.1 for EcoRI is also suitable for T4 DNA polymerase and, 1ug DNA ~ 1 unit enzyme; final concentration for dNTP: 100uM]
-
for T4 DNA polymerase and, </span>
+
-
<span lang="EN-US">1ug DNA ~ 1 unit enzyme; final concentration for dNTP: 100uM]</span>
+
'''3. Protocol:'''
-
'''<span lang="EN-US">3. Protocol:</span>'''
+
a. incubate the fill-in system at 12°C，15min
-
<span lang="EN-US">a. incubate the fill-in system at 12</span>°<span lang="EN-US">C</span>，<span lang="EN-US">15min</span>
+
b. add 2ul EDTA(100mM) to 18ul reaction
 +
system to stop the polyreaction.
-
<span lang="EN-US">b. add 2ul EDTA(100mM) to 18ul reaction
+
c. put in 65°C，20min to
-
system to stop the polyreaction. </span>
+
inactivate T4 DNA polymerase thoroughly.
-
<span lang="EN-US">c. put in 65</span>°<span lang="EN-US">C</span>，<span lang="EN-US">20min to
+
'''4. Accident：'''
-
inactivate T4 DNA polymerase thoroughly.</span>
+
-
'''<span lang="EN-US">4. Accident</span>：'''
+
To begin with, due to our carelessness, we
-
 
+
-
<span lang="EN-US">To begin with, due to our carelessness, we
+
added Taq DNA polymerase instead of T4 DNA polymerase to the 19ul system. But,
added Taq DNA polymerase instead of T4 DNA polymerase to the 19ul system. But,
thinking that it might not cause any serious consequence, we then just added T4
thinking that it might not cause any serious consequence, we then just added T4
-
DNA polymerase into the system with the same volume.</span>
+
DNA polymerase into the system with the same volume.
-
'''<span lang="EN-US">5. Electrophoresis & Agrose gel DNA extraction to remove enzymes and competent of Buffer</span>'''
+
===Electrophoresis & Agrose gel DNA extraction to remove enzymes and competent of Buffer===
-
'''<span lang="EN-US">DNA extraction:</span>'''
+
'''DNA extraction:'''
-
<span lang="EN-US">Performed as the protocol provided by
+
Performed as the protocol provided by
TIANgel Purification Kit, except that we eluted the DNA sample twice at the
TIANgel Purification Kit, except that we eluted the DNA sample twice at the
-
last step (as we always done before). </span>
+
last step (as we always done before).  
-
'''<span lang="EN-US">Concentration of the purified DNA fragments:</span>'''
+
'''Concentration of the purified DNA fragments:'''
-
<span lang="EN-US">Poly A: 19.3ng/ul</span>
+
Poly A: 19.3ng/ul
-
<span lang="EN-US">G-PB5: 18.8ng/ul</span>
+
G-PB5: 18.8ng/ul
-
'''<span lang="EN-US">6. Blunt end ligation with
+
===Blunt end ligation with T4 DNA ligase [Design a concentration gradient]===
-
T4 DNA ligase [Design a concentration gradient]</span>'''
+
-
'''<span lang="EN-US">1) Ligation system</span>：'''（<span lang="EN-US">unit: ul</span>）
+
'''1) Ligation system：'''（unit: ul）
-
{|
+
{|class="table"
-
+
|
-
  |
+
|Total  volume
-
<nowiki>  </nowiki><span lang="EN-US"> </span>
+
|DNA
-
 
+
|10X  T4 DNA ligase buffer
-
  |
+
|T4  ligase
-
<nowiki>  </nowiki><span lang="EN-US">Total  volume</span>
+
|EcoRI (to reduce those sticky  ends ligation)
-
 
+
|ddH2O
-
  |
+
|-
-
<nowiki>  </nowiki><span lang="EN-US">DNA</span>
+
|(40ng rank) 1.2 tube
-
 
+
|21
-
  |
+
|2
-
<nowiki>  </nowiki><span lang="EN-US">10X  T4 DNA ligase buffer</span>
+
|2
-
 
+
|1
-
  |
+
|1
-
<nowiki>  </nowiki><span lang="EN-US">T4  ligase</span>
+
|15
-
 
+
|-
-
  |
+
|(100ng rank) 3,4 tube
-
<nowiki>  </nowiki><span lang="EN-US">EcoRI</span>
+
|21
-
 
+
|6
-
<span lang="EN-US">(to reduce those sticky  ends ligation)</span>
+
|2
-
 
+
|2
-
  |
+
|1
-
<nowiki>  </nowiki><span lang="EN-US">ddH2O</span> 
+
|10
-
+
|-
-
|-
+
|(200ng rank) 5,6 tube
-
  |
+
|21
-
<nowiki>  </nowiki><span lang="EN-US">(40ng rank)</span>
+
|12
-
 
+
|2
-
<span lang="EN-US">1.2 tube</span>
+
|2
-
 
+
|1
-
  |
+
|4
-
<nowiki>  </nowiki><span lang="EN-US">21</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">2</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">2</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">1</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">1</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">15</span> 
+
-
+
-
|-
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">(100ng rank)</span>
+
-
 
+
-
<span lang="EN-US">3,4 tube</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">21</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">6</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">2</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">2</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">1</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">10</span> 
+
-
+
-
|-
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">(200ng rank)</span>
+
-
 
+
-
<span lang="EN-US">5,6 tube</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">21</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">12</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">2</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">2</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">1</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">4</span> 
+
-
+
|}
|}
-
'''<span lang="EN-US">2) Protocol</span>：'''
 
-
<span lang="EN-US">a. incubate the ligation system at 16</span>°<span lang="EN-US">C</span>，<span lang="EN-US">15h [8.9
+
'''2) Protocol：'''
-
1:30pm ~ 8.10 4:30am ]</span>
+
-
<span lang="EN-US">b. store at 4</span>°<span lang="EN-US">C [8.10 4:30am ~10:00am]</span>
+
a. incubate the ligation system at 16°C，15h [8.9
 +
1:30pm ~ 8.10 4:30am ]
-
<span lang="EN-US">c. Heat inactivation, 65</span>°<span lang="EN-US">C, 10min</span>
+
b. store at 4°C [8.10 4:30am ~10:00am]
-
<span lang="EN-US">d. chill on ice</span>
+
c. Heat inactivation, 65°C, 10min
-
'''<span lang="EN-US">7. Electrophoresis &
+
d. chill on ice
-
Agrose gel DNA extraction to remove enzymes and competent of Buffer</span>'''
+
-
'''<span lang="EN-US">DNA extraction:</span>'''
+
===Electrophoresis & Agrose gel DNA extraction to remove enzymes and competent of Buffer===
-
<span lang="EN-US">Performed as the protocol… but, we forgot
+
'''DNA extraction:'''
 +
 
 +
Performed as the protocol… but, we forgot
to change the collecting tubes into EP tubes before adding EB solution…So, the
to change the collecting tubes into EP tubes before adding EB solution…So, the
most of the DNA was eluted into collecting tubes which might also contain many
most of the DNA was eluted into collecting tubes which might also contain many
-
impurities(like ethanol, proteins…).</span>
+
impurities(like ethanol, proteins…).
-
<span lang="EN-US"> </span>
+
 
-
<span lang="EN-US">Anyway, we still transferred DNA in the
+
Anyway, we still transferred DNA in the
-
collecting tubes into new EP tubes, and then detect the concentration of them.</span>
+
collecting tubes into new EP tubes, and then detect the concentration of them.
-
{|
+
{|class="table"
-
+
|
-
  |
+
|Concentration  (ng/ul)
-
<nowiki>  </nowiki><span lang="EN-US"> </span>
+
|260/280
-
 
+
|260/230   
-
  |
+
|-
-
<nowiki>  </nowiki><span lang="EN-US">Concentration  (ng/ul)</span>
+
|G-PB5 1,2
-
 
+
|24.6
-
  |
+
|6.83
-
<nowiki>  </nowiki><span lang="EN-US">260/280</span>
+
|0.07  
-
 
+
|-
-
  |
+
|G-PB5 3,4
-
<nowiki>  </nowiki><span lang="EN-US">260/230</span>  
+
|17.5
-
+
|7.43
-
|-
+
|0.04
-
  |
+
|-
-
<nowiki>  </nowiki><span lang="EN-US">G-PB5 1,2</span>
+
|G-PB5 5,6
-
 
+
|31.1
-
  |
+
|5.42
-
<nowiki>  </nowiki><span lang="EN-US">24.6</span>
+
|0.06   
-
 
+
|-
-
  |
+
|Poly A 1,2
-
<nowiki>  </nowiki><span lang="EN-US">6.83</span>
+
|21.4
-
 
+
|5.07
-
  |
+
|0.06
-
<nowiki>  </nowiki><span lang="EN-US">0.07</span> 
+
|-
-
+
|Poly A 3,4
-
|-
+
|23.5
-
  |
+
|6.31
-
<nowiki>  </nowiki><span lang="EN-US">G-PB5 3,4</span>
+
|0.05
-
 
+
|-
-
  |
+
|Poly A 5,6
-
<nowiki>  </nowiki><span lang="EN-US">17.5</span>
+
|25.8
-
 
+
|5.44
-
  |
+
|0.06
-
<nowiki>  </nowiki><span lang="EN-US">7.43</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">0.04</span> 
+
-
+
-
|-
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">G-PB5 5,6</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">31.1</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">5.42</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">0.06</span>  
+
-
+
-
|-
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">Poly A 1,2</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">21.4</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">5.07</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">0.06</span> 
+
-
+
-
|-
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">Poly A 3,4</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">23.5</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">6.31</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">0.05</span> 
+
-
+
-
|-
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">Poly A 5,6</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">25.8</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">5.44</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">0.06</span> 
+
-
+
|}
|}
-
<span lang="EN-US">Although everything seems normal, but we
+
Although everything seems normal, but we
-
still decided not to use them.</span>
+
still decided not to use them.
-
<span lang="EN-US"> </span>
+
 
-
<span lang="EN-US">To save our 2 day’s endeavor, we then put
+
To save our 2 day’s endeavor, we then put
CA2 tubes into new EP tubes, and add 20ul EB solution, wishing to elute some
CA2 tubes into new EP tubes, and add 20ul EB solution, wishing to elute some
DNA that haven’t been washed off for the first time. Fortunately, after
DNA that haven’t been washed off for the first time. Fortunately, after
-
two-time elution, we finally got relatively pure DNA samples.</span>
+
two-time elution, we finally got relatively pure DNA samples.
-
{|
+
{|class="table"
-
+
|
-
  |
+
|Concentration  (ng/ul)
-
<nowiki>  </nowiki><span lang="EN-US"> </span>
+
|260/280
-
 
+
|260/230   
-
  |
+
|-
-
<nowiki>  </nowiki><span lang="EN-US">Concentration  (ng/ul)</span>
+
|G-PB5 1,2
-
 
+
|29.8
-
  |
+
|8.05
-
<nowiki>  </nowiki><span lang="EN-US">260/280</span>
+
|0.09
-
 
+
|-
-
  |
+
|G-PB5 3,4
-
<nowiki>  </nowiki><span lang="EN-US">260/230</span>  
+
|22.1
-
+
|9.53
-
|-
+
|0.07
-
  |
+
|-
-
<nowiki>  </nowiki><span lang="EN-US">G-PB5 1,2</span>
+
|G-PB5 5,6
-
 
+
|29.7
-
  |
+
|7.01
-
<nowiki>  </nowiki><span lang="EN-US">29.8</span>
+
|0.09
-
 
+
|-
-
  |
+
|Poly A 1,2
-
<nowiki>  </nowiki><span lang="EN-US">8.05</span>
+
|22.9
-
 
+
|11.53
-
  |
+
|0.07
-
<nowiki>  </nowiki><span lang="EN-US">0.09</span> 
+
|-
-
+
|Poly A 3,4
-
|-
+
|22.2
-
  |
+
|8.24
-
<nowiki>  </nowiki><span lang="EN-US">G-PB5 3,4</span>
+
|0.07  
-
 
+
|-
-
  |
+
|Poly A 5,6
-
<nowiki>  </nowiki><span lang="EN-US">22.1</span>
+
|19.9
-
 
+
|11.63
-
  |
+
|0.06
-
<nowiki>  </nowiki><span lang="EN-US">9.53</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">0.07</span> 
+
-
+
-
|-
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">G-PB5 5,6</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">29.7</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">7.01</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">0.09</span> 
+
-
+
-
|-
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">Poly A 1,2</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">22.9</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">11.53</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">0.07</span> 
+
-
+
-
|-
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">Poly A 3,4</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">22.2</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">8.24</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">0.07</span> 
+
-
+
-
|-
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">Poly A 5,6</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">19.9</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">11.63</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">0.06</span> 
+
-
+
|}
|}
-
<span lang="EN-US"> </span>
+
 
-
 
+
One abnormal phenomenon is that, in our ligation system, the total amount of DNA is 200ng or so at most. But from our extraction results, we can see that the quantity of DNA in each tube reaches 400ng around. So, where did these extra DNA come from? Or, is it just caused by the inaccurate measurement of Nanodrop?
-
'''''<span lang="EN-US">One</span>'''<span lang="EN-US"> abnormal phenomenon is that, in
+
-
our ligation system, the total amount of DNA is 200ng or so at most. But from
+
-
our extraction results, we can see that the quantity of DNA in each tube reaches 400ng around. So, where did these extra DNA come from? Or, is it just caused by the inaccurate measurement of Nanodrop?</span>''
+
-
'''<span lang="EN-US">8. Enzyme Digestion for recovered DNA, to relinearize those sticky-end self-ligations</span>'''
+
===Enzyme Digestion for recovered DNA, to relinearize those sticky-end self-ligations===
-
'''<span lang="EN-US">1) Digestion system</span>'''<span lang="EN-US"> [unit: ul]</span>
+
'''1) Digestion system''' [unit: ul]
-
{|
+
{|class="table"
-
+
|Total  volume
-
  |
+
|DNA
-
<nowiki>  </nowiki><span lang="EN-US">Total  volume</span>
+
|EcoRI
-
 
+
|10X NEBuffer
-
  |
+
|-
-
<nowiki>  </nowiki><span lang="EN-US">DNA</span>
+
|10
-
 
+
|8.5
-
  |
+
|0.5
-
<nowiki>  </nowiki><span lang="EN-US">EcoRI</span>
+
|1
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">10X
+
-
  NEBuffer</span> 
+
-
+
-
|-
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">10</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">8.5</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">0.5</span>
+
-
 
+
-
  |
+
-
<nowiki>  </nowiki><span lang="EN-US">1</span> 
+
-
+
|}
|}
-
'''<span lang="EN-US">2)
+
'''2) Reserve the rest 10ul DNA as backups'''
-
Reserve the rest 10ul DNA as backups</span>'''
+
-
'''<span lang="EN-US">3)
+
'''3) incubate the digestion system''' 37°C， 4h [12:35am ~ 5:00am]
-
incubate the digestion system</span>'''<span lang="EN-US">  37</span>°<span lang="EN-US">C</span>，<span lang="EN-US"> 4h [12:35am
+
-
~ 5:00am]</span>
+
-
'''<span lang="EN-US">9. Transformation</span>'''
+
'''9. Transformation'''
-
''<span lang="EN-US">Performed as the usual protocol</span>''
+
''Performed as the usual protocol''
-
<span lang="EN-US">1. Add 5ul DNA into 50ul competent cell. [Save the remaining 5ul DNA as backups]</span>
+
1. Add 5ul DNA into 50ul competent cell. [Save the remaining 5ul DNA as backups]
-
<span lang="EN-US">2. We also transformed bacteria with those
+
2. We also transformed bacteria with those
-
DNA that haven’t been digested by EcoRI as well. To make sure, if there were no colonies growing out, the reason shouldn’t lie in the ligation step.</span>
+
DNA that haven’t been digested by EcoRI as well. To make sure, if there were no colonies growing out, the reason shouldn’t lie in the ligation step.
-
<span lang="EN-US">3. Recover step: 200rpm, 45min, 18:44pm~19:30pm</span>
+
3. Recover step: 200rpm, 45min, 18:44pm~19:30pm
-
<span lang="EN-US">4. Centrifuge before distributing on agar plate: 4000rpm, 5min</span>
+
4. Centrifuge before distributing on agar plate: 4000rpm, 5min
-
<span lang="EN-US">5. Incubate, 37</span>°<span lang="EN-US">C, overnight [2014.8.10 8:44pm~2014.8.11 10:00am]</span>
+
5. Incubate, 37°C, overnight [2014.8.10 8:44pm~2014.8.11 10:00am]
-
'''<span lang="EN-US">Results</span>：'''
+
==Results==
-
'''<span lang="EN-US">Digested groups:</span>'''
+
===Digested groups===
-
<span lang="EN-US">Except G1,2 group with digested plasmid have colonies grown out, all the other
+
Except G1,2 group with digested plasmid have colonies grown out, all the other digested groups have no colonies grown on. However, the colonies that grown on the G1,2 digested plates looked extremely abnormal. There are too many colonies growing on the plate that we nearly cannot distinguish any monocolones. Such strange result implied the failure of our second-time Fill-in.
-
digested groups have no colonies grown on. However, the colonies that grown on
+
-
the G1,2 digested plates looked extremely abnormal. There are too many colonies
+
-
growing on the plate that we nearly cannot distinguish any monocolones. Such strange result implied the failure of our second-time Fill-in.</span>
+
-
'''<span lang="EN-US">Undigested groups:</span>'''
+
===Undigested groups===
-
<span lang="EN-US">Some
+
Some groups have colonies grown out on their undigested plates which can roughly
-
groups have colonies grown out on their undigested plates which can roughly
+
indicate that our ligation was operative.  
-
indicate that our ligation was operative. </span>
+
-
<span lang="EN-US"> </span>
+
 
-
'''<span lang="EN-US">Improvement:</span>'''
+
===Improvement===
-
<span lang="EN-US">1) Increase
+
1) Increase the quantity of DNA in each system  
-
the quantity of DNA in each system </span>
+
-
<span lang="EN-US">2) Heat
+
2) Heat inactivation before adding T4 DNA polymerase to avoid interplays.
-
inactivation before adding T4 DNA polymerase to avoid interplays.</span>
+
-
<span lang="EN-US">3)
+
3) Perform restriction digestion directly after ligation step without DNA extraction.
-
Perform restriction digestion directly after ligation step without DNA
+
-
extraction.</span>
+
-
<span lang="EN-US">4) If we failed again for the third time,
+
4) If we failed again for the third time, we plan to apply new method to eliminate the EcoRI site on UAS sequence. Such as, site-specific mutagenesis, or isocaudarner ligation.
-
we plan to apply new method to eliminate the EcoRI site on UAS sequence. Such
+
-
as, site-specific mutagenesis, or isocaudarner ligation.</span>[[Category:VisualEditor]]*
+

Latest revision as of 13:42, 17 October 2014

Team SUSTC-Shenzhen

Notebook

Elements of the endeavor.

UAS Fill-in (The second time)

2014/8/9


Materials

The same as the first Fill-in experiment.

Methods

Restriction digestion for 5*UAS plasmid with EcoRI (unit: ul)

Total volume 10X Buffer DNA EcoRI ddH2O
Poly A(168.8ng/ul) 20 2 2.5 1 14.5
G-PB5(293.6ng/ul) 20 2 2 1 15

Time: 2014.8.8 10:00pm ~ 2014.8.9 9:30am (Incubate overnight)

Electrophoresis to verify if most of the digested plasmid has been linearized

Loading system：(unit: ul)

Total volume/well DNA Dye TAE
12 0.5 2 9.5

Running conditions: 130V,   30min

Results of electrophoresis:

{{{2}}}

From the picture, we can see that, the position of the band of digested plasmid is apparently lying behind that of the original plasmid which hasn’t been digested by EcoRI, indicating that most of the plasmid in our digestion system has become linearized.

Heat-inactivation for the digestion system & Cold shock to prevent self-ligation

1. Heat-inactivation 75°C，10min

2. Chill on ice, 15min

Do fill-in with T4 DNA polymerase

1. Discard 0.5ul DNA from the former inactivated digestion system, forming the final 19ul system.

Till now, the concentration of DNA for:

Poly A: 21.1ng/ul

G-PB5: 29.36ng/ul

2. Fill-insystem：(unit: ul)

Total volume DNA T4 DNA polymerase(3U/ul) dNTP (10mM) ddH2O
Poly A(21.1ng/ul) 20 19 0.2 (theoretical volume:0.13ul) 0.2 0.6
G-PB5(29.36ng/ul) 20 19 0.2 (theoretical volume:0.186ul) 0.2 0.6

[According to the manual provided by NEB：The NEBuffer 2.1 for EcoRI is also suitable for T4 DNA polymerase and, 1ug DNA ~ 1 unit enzyme; final concentration for dNTP: 100uM]

3. Protocol:

a. incubate the fill-in system at 12°C，15min

b. add 2ul EDTA(100mM) to 18ul reaction system to stop the polyreaction.

c. put in 65°C，20min to inactivate T4 DNA polymerase thoroughly.

4. Accident：

To begin with, due to our carelessness, we added Taq DNA polymerase instead of T4 DNA polymerase to the 19ul system. But, thinking that it might not cause any serious consequence, we then just added T4 DNA polymerase into the system with the same volume.

Electrophoresis & Agrose gel DNA extraction to remove enzymes and competent of Buffer

DNA extraction:

Performed as the protocol provided by TIANgel Purification Kit, except that we eluted the DNA sample twice at the last step (as we always done before).

Concentration of the purified DNA fragments:

Poly A: 19.3ng/ul

G-PB5: 18.8ng/ul

Blunt end ligation with T4 DNA ligase [Design a concentration gradient]

1) Ligation system：（unit: ul）

Total volume DNA 10X T4 DNA ligase buffer T4 ligase EcoRI (to reduce those sticky ends ligation) ddH2O
(40ng rank) 1.2 tube 21 2 2 1 1 15
(100ng rank) 3,4 tube 21 6 2 2 1 10
(200ng rank) 5,6 tube 21 12 2 2 1 4

2) Protocol：

a. incubate the ligation system at 16°C，15h [8.9 1:30pm ~ 8.10 4:30am ]

b. store at 4°C [8.10 4:30am ~10:00am]

c. Heat inactivation, 65°C, 10min

d. chill on ice

Electrophoresis & Agrose gel DNA extraction to remove enzymes and competent of Buffer

DNA extraction:

Performed as the protocol… but, we forgot to change the collecting tubes into EP tubes before adding EB solution…So, the most of the DNA was eluted into collecting tubes which might also contain many impurities(like ethanol, proteins…).

 

Anyway, we still transferred DNA in the collecting tubes into new EP tubes, and then detect the concentration of them.

Concentration (ng/ul) 260/280 260/230
G-PB5 1,2 24.6 6.83 0.07
G-PB5 3,4 17.5 7.43 0.04
G-PB5 5,6 31.1 5.42 0.06
Poly A 1,2 21.4 5.07 0.06
Poly A 3,4 23.5 6.31 0.05
Poly A 5,6 25.8 5.44 0.06

Although everything seems normal, but we still decided not to use them.

 

To save our 2 day’s endeavor, we then put CA2 tubes into new EP tubes, and add 20ul EB solution, wishing to elute some DNA that haven’t been washed off for the first time. Fortunately, after two-time elution, we finally got relatively pure DNA samples.

Concentration (ng/ul) 260/280 260/230
G-PB5 1,2 29.8 8.05 0.09
G-PB5 3,4 22.1 9.53 0.07
G-PB5 5,6 29.7 7.01 0.09
Poly A 1,2 22.9 11.53 0.07
Poly A 3,4 22.2 8.24 0.07
Poly A 5,6 19.9 11.63 0.06

  One abnormal phenomenon is that, in our ligation system, the total amount of DNA is 200ng or so at most. But from our extraction results, we can see that the quantity of DNA in each tube reaches 400ng around. So, where did these extra DNA come from? Or, is it just caused by the inaccurate measurement of Nanodrop?

Enzyme Digestion for recovered DNA, to relinearize those sticky-end self-ligations

1) Digestion system [unit: ul]

Total volume DNA EcoRI 10X NEBuffer
10 8.5 0.5 1

2) Reserve the rest 10ul DNA as backups

3) incubate the digestion system 37°C， 4h [12:35am ~ 5:00am]

9. Transformation

Performed as the usual protocol

1. Add 5ul DNA into 50ul competent cell. [Save the remaining 5ul DNA as backups]

2. We also transformed bacteria with those DNA that haven’t been digested by EcoRI as well. To make sure, if there were no colonies growing out, the reason shouldn’t lie in the ligation step.

3. Recover step: 200rpm, 45min, 18:44pm~19:30pm

4. Centrifuge before distributing on agar plate: 4000rpm, 5min

5. Incubate, 37°C, overnight [2014.8.10 8:44pm~2014.8.11 10:00am]

Results

Digested groups

Except G1,2 group with digested plasmid have colonies grown out, all the other digested groups have no colonies grown on. However, the colonies that grown on the G1,2 digested plates looked extremely abnormal. There are too many colonies growing on the plate that we nearly cannot distinguish any monocolones. Such strange result implied the failure of our second-time Fill-in.

Undigested groups

Some groups have colonies grown out on their undigested plates which can roughly indicate that our ligation was operative.

 

Improvement

1) Increase the quantity of DNA in each system

2) Heat inactivation before adding T4 DNA polymerase to avoid interplays.

3) Perform restriction digestion directly after ligation step without DNA extraction.

4) If we failed again for the third time, we plan to apply new method to eliminate the EcoRI site on UAS sequence. Such as, site-specific mutagenesis, or isocaudarner ligation.


Maintained by the iGEM team SUSTC-Shenzhen.

Licensed under CC BY 4.0.

  • ©2014 SUSTC-Shenzhen

Retrieved from "http://2014.igem.org/Team:SUSTC-Shenzhen/Notebook/CRISPR/UAS-Fill-in-second-time"