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

From 2014.igem.org

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<span lang="EN-US">Materials</span>
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<span lang="EN-US">The same as the first Fill-in experiment</span>
-
      title=Notebook|
+
-
      subtitle=Elements of the endeavor.}}
+
-
{{SUSTC-Shenzhen/main-content-begin}}
+
'''<span lang="EN-US">Methods:</span>'''
-
{{:Team:SUSTC-Shenzhen/templates/notebook-main|
+
'''<span lang="EN-US">1. Restriction digestion for 5*UAS plasmid with EcoRI</span>'''（<span lang="EN-US">unit: ul</span>）
-
      name=UAS Fill-in (The second time)|
+
{|
-
      date=2014/8/9|
+
-
      goal=The same as the first Fill-in experiment}}
+
  |
 +
<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>
-
==Materials==
+
'''<span lang="EN-US">2. Electrophoresis to verify
-
The same as the first Fill-in experiment
+
if most of the digested plasmid has been linearized</span>'''
-
==Methods==
+
'''<span lang="EN-US">Loading system</span>：'''(<span lang="EN-US">unit: ul</span>)
-
1. Restriction digestion for 5*UAS plasmid with EcoRI（unit: ul）
+
{|
-
{| class="table"
+
-
|-
+
  |
-
! !! Total volume !! 10X Buffer !! DNA !! EcoRI !! ddH2O
+
<nowiki>  </nowiki><span lang="EN-US">Total  volume/well</span>
-
|-
+
 
-
| Poly A(168.8ng/ul) || 20 || 2 || 2.5 || 1 || 14.5  
+
  |
-
|-
+
<nowiki> </nowiki><span lang="EN-US">DNA</span>
-
| G-PB5(293.6ng/ul) || 20 || 2 || 2 || 1 || 15
+
 
 +
  |
 +
<nowiki>  </nowiki><span lang="EN-US">Dye</span>
 +
 
 +
  |
 +
<nowiki>  </nowiki><span lang="EN-US">TAE</span> 
 +
 +
|-
 +
  |
 +
<nowiki>  </nowiki><span lang="EN-US">12</span>
 +
 
 +
  |
 +
<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>
-
Time: 2014.8.8 10:00pm ~ 2014.8.9 9:30am ( Incubate overnight)
+
'''<span lang="EN-US">Results of electrophoresis:</span>'''
-
2. Electrophoresis to verify if most of the digested plasmid has been linearized
+
<span lang="EN-US"> </span>
-
Loading system：（unit: ul）
+
 
-
{| class="table"
+
<span lang="EN-US"> </span>
-
|-
+
 
-
! Total volume/well !! DNA !! Dye !! TAE
+
<span lang="EN-US"> </span>
-
|-
+
 
-
| 12 || 0.5 || 2 || 9.5
+
<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"> </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 original plasmid which hasn’t been digested by EcoRI, indicating that most
 +
of the plasmid in our digestion system has become linearized. </span>
 +
 
 +
'''<span lang="EN-US">3. Heat-inactivation for the
 +
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>
 +
 
 +
<span lang="EN-US">2. Chill on ice, 15min</span>
 +
 
 +
'''<span lang="EN-US">4. Do fill-in with T4 DNA
 +
polymerase</span>'''
 +
 
 +
'''<span lang="EN-US">1.
 +
Discard 0.5ul DNA from the former inactivated digestion system, forming the
 +
final 19ul system.</span>'''
 +
 
 +
<span lang="EN-US">Till now, the concentration of DNA for:</span>
 +
 
 +
<span lang="EN-US">Poly A: 21.1ng/ul</span>
 +
 
 +
<span lang="EN-US">            G-PB5: 29.36ng/ul</span>
 +
 
 +
'''<span lang="EN-US">2. Fill-insystem</span>：'''（<span lang="EN-US">unit: ul</span>）
 +
{|
 +
 +
  |
 +
<nowiki>  </nowiki><span lang="EN-US"> </span>
 +
 
 +
  |
 +
<nowiki>  </nowiki><span lang="EN-US">Total volume</span>
 +
 
 +
  |
 +
<nowiki>  </nowiki><span lang="EN-US">DNA</span>
 +
 
 +
  |
 +
<nowiki>  </nowiki><span lang="EN-US">T4
 +
  DNA polymerase(3U/ul)</span>
 +
 
 +
  |
 +
<nowiki>  </nowiki><span lang="EN-US">dNTP  (10mM)</span>
 +
 
 +
  |
 +
<nowiki>  </nowiki><span lang="EN-US">ddH2O</span> 
 +
 +
|-
 +
  |
 +
<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> 
 +
|}
|}
-
Running conditions: 130V,  30min
+
<span lang="EN-US">[According to the manual provided by NEB</span>：
-
Results of electrophoresis:
+
 +
<span lang="EN-US">The NEBuffer 2.1 for EcoRI is also suitable
 +
for T4 DNA polymerase and, </span>
 +
<span lang="EN-US">1ug DNA ~ 1 unit enzyme; final concentration for dNTP: 100uM]</span>
 +
'''<span lang="EN-US">3. Protocol:</span>'''
 +
<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>
 +
<span lang="EN-US">b. add 2ul EDTA(100mM) to 18ul reaction
 +
system to stop the polyreaction. </span>
 +
<span lang="EN-US">c. put in 65</span>°<span lang="EN-US">C</span>，<span lang="EN-US">20min to
 +
inactivate T4 DNA polymerase thoroughly.</span>
 +
'''<span lang="EN-US">4. Accident</span>：'''
 +
<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,
 +
thinking that it might not cause any serious consequence, we then just added T4
 +
DNA polymerase into the system with the same volume.</span>
 +
'''<span lang="EN-US">5. Electrophoresis & Agrose gel DNA extraction to remove enzymes and competent of Buffer</span>'''
 +
'''<span lang="EN-US">DNA extraction:</span>'''
 +
<span lang="EN-US">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). </span>
 +
'''<span lang="EN-US">Concentration of the purified DNA fragments:</span>'''
-
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.  
+
<span lang="EN-US">Poly A: 19.3ng/ul</span>
-
3. Heat-inactivation for the digestion system & Cold shock to prevent self-ligation
+
<span lang="EN-US">G-PB5: 18.8ng/ul</span>
-
# Heat-inactivation 75°C，10min
+
 
-
# Chill on ice, 15min
+
'''<span lang="EN-US">6. Blunt end ligation with
-
4. Do fill-in with T4 DNA polymerase
+
T4 DNA ligase [Design a concentration gradient]</span>'''
-
# Discard 0.5ul DNA from the former inactivated digestion system, forming the final 19ul system.
+
 
-
#:Till now, the concentration of DNA for:
+
'''<span lang="EN-US">1) Ligation system</span>：'''（<span lang="EN-US">unit: ul</span>）
-
#:: Poly A: 21.1ng/ul
+
{|
-
#:: G-PB5: 29.36ng/ul
+
-
# Fill-insystem：（unit: ul）
+
  |
-
{| class="table"
+
<nowiki>  </nowiki><span lang="EN-US"> </span>
-
!
+
 
-
! Total
+
  |
-
   volume
+
<nowiki>  </nowiki><span lang="EN-US">Total volume</span>
-
! DNA
+
    
-
! T4
+
  |
-
   DNA polymerase(3U/ul)
+
<nowiki>  </nowiki><span lang="EN-US">DNA</span>
-
! dNTP
+
 
-
   (10mM)
+
   |
-
! ddH2O
+
<nowiki>  </nowiki><span lang="EN-US">10X  T4 DNA ligase buffer</span>
-
|-
+
 
-
| Poly
+
   |
-
   A(21.1ng/ul)
+
<nowiki>  </nowiki><span lang="EN-US">T4  ligase</span>
-
| 20
+
 
-
| 19
+
  |
-
| 0.2
+
<nowiki>  </nowiki><span lang="EN-US">EcoRI</span>
-
   (theoretical volume:0.13ul)
+
 
-
| 0.2
+
<span lang="EN-US">(to reduce those sticky  ends ligation)</span>
-
| 0.6
+
 
-
|-
+
  |
-
| G-PB5(29.36ng/ul)
+
<nowiki>  </nowiki><span lang="EN-US">ddH2O</span> 
-
| 20
+
-
| 19
+
|-
-
| 0.2
+
   |
-
   (theoretical
+
<nowiki>  </nowiki><span lang="EN-US">(40ng rank)</span>
-
   volume:0.186ul)
+
 
-
| 0.2
+
<span lang="EN-US">1.2 tube</span>
-
| 0.6
+
 
 +
  |
 +
<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> 
 +
|}
|}
-
[According to the manual provided by NEB：
+
'''<span lang="EN-US">2) Protocol</span>：'''
-
The NEBuffer 2.1 for EcoRI is also suitable for T4 DNA polymerase and,
+
 
-
1ug DNA ~ 1 unit enzyme; final concentration for dNTP: 100uM]
+
<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
-
# Protocol:
+
1:30pm ~ 8.10 4:30am ]</span>
-
## incubate the fill-in system at 12°C，15min
+
 
-
## add 2ul EDTA(100mM) to 18ul reaction system to stop the polyreaction.  
+
<span lang="EN-US">b. store at 4</span>°<span lang="EN-US">C [8.10 4:30am ~10:00am]</span>
-
## put in 65°C，20min to inactivate T4 DNA polymerase thoroughly.
+
 
-
#Accident：
+
<span lang="EN-US">c. Heat inactivation, 65</span>°<span lang="EN-US">C, 10min</span>
-
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.
+
 
-
5. Electrophoresis & Agrose gel DNA extraction to remove enzymes and competent of Buffer
+
<span lang="EN-US">d. chill on ice</span>
-
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).  
+
'''<span lang="EN-US">7. Electrophoresis &
-
Concentration of the purified DNA fragments:
+
Agrose gel DNA extraction to remove enzymes and competent of Buffer</span>'''
-
Poly A: 19.3ng/ul
+
 
-
G-PB5: 18.8ng/ul
+
'''<span lang="EN-US">DNA extraction:</span>'''
-
6. Blunt end ligation with T4 DNA ligase [Design a concentration gradient]
+
 
-
1) Ligation system：（unit: ul）
+
<span lang="EN-US">Performed as the protocol… but, we forgot
-
Total volume DNA 10X T4 DNA ligase buffer T4 ligase EcoRI
+
to change the collecting tubes into EP tubes before adding EB solution…So, the
-
(to reduce those sticky ends ligation) ddH2O
+
most of the DNA was eluted into collecting tubes which might also contain many
-
(40ng rank)
+
impurities(like ethanol, proteins…).</span>
-
1.2 tube 21 2 2 1 1 15
+
 
-
(100ng rank)
+
<span lang="EN-US"> </span>
-
3,4 tube 21 6 2 2 1 10
+
 
-
(200ng rank)
+
<span lang="EN-US">Anyway, we still transferred DNA in the
-
5,6 tube 21 12 2 2 1 4
+
collecting tubes into new EP tubes, and then detect the concentration of them.</span>
-
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
+
<nowiki>  </nowiki><span lang="EN-US"> </span>
-
d. chill on ice
+
 
-
7. Electrophoresis & Agrose gel DNA extraction to remove enzymes and competent of Buffer
+
  |
-
DNA extraction:
+
<nowiki>  </nowiki><span lang="EN-US">Concentration  (ng/ul)</span>
-
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…).
+
 
 +
  |
 +
<nowiki>  </nowiki><span lang="EN-US">260/280</span>
 +
 
 +
  |
 +
<nowiki>  </nowiki><span lang="EN-US">260/230</span> 
 +
 +
|-
 +
  |
 +
<nowiki>  </nowiki><span lang="EN-US">G-PB5 1,2</span>
 +
 
 +
  |
 +
<nowiki>  </nowiki><span lang="EN-US">24.6</span>
 +
 
 +
  |
 +
<nowiki>  </nowiki><span lang="EN-US">6.83</span>
 +
 
 +
  |
 +
<nowiki>  </nowiki><span lang="EN-US">0.07</span> 
 +
 +
|-
 +
  |
 +
<nowiki>  </nowiki><span lang="EN-US">G-PB5  3,4</span>
 +
 
 +
  |
 +
<nowiki>  </nowiki><span lang="EN-US">17.5</span>
 +
 
 +
  |
 +
<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
 +
still decided not to use them.</span>
 +
 
 +
<span lang="EN-US"> </span>
 +
 
 +
<span lang="EN-US">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.</span>
 +
{|
 +
 +
  |
 +
<nowiki>  </nowiki><span lang="EN-US"> </span>
 +
 
 +
  |
 +
<nowiki>  </nowiki><span lang="EN-US">Concentration  (ng/ul)</span>
 +
 
 +
  |
 +
<nowiki>  </nowiki><span lang="EN-US">260/280</span>
 +
 
 +
  |
 +
<nowiki>  </nowiki><span lang="EN-US">260/230</span> 
 +
 +
|-
 +
  |
 +
<nowiki>  </nowiki><span lang="EN-US">G-PB5  1,2</span>
 +
 
 +
  |
 +
<nowiki>  </nowiki><span lang="EN-US">29.8</span>
 +
 
 +
  |
 +
<nowiki>  </nowiki><span lang="EN-US">8.05</span>
 +
 
 +
  |
 +
<nowiki>  </nowiki><span lang="EN-US">0.09</span> 
 +
 +
|-
 +
  |
 +
<nowiki>  </nowiki><span lang="EN-US">G-PB5  3,4</span>
 +
 
 +
  |
 +
<nowiki>  </nowiki><span lang="EN-US">22.1</span>
 +
 
 +
  |
 +
<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>
 +
 
 +
'''''<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>'''
 +
 
 +
'''<span lang="EN-US">1) Digestion system</span>'''<span lang="EN-US"> [unit: ul]</span>
 +
{|
 +
 +
  |
 +
<nowiki>  </nowiki><span lang="EN-US">Total  volume</span>
 +
 
 +
  |
 +
<nowiki>  </nowiki><span lang="EN-US">DNA</span>
 +
 
 +
  |
 +
<nowiki>  </nowiki><span lang="EN-US">EcoRI</span>
 +
 
 +
  |
 +
<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)
 +
Reserve the rest 10ul DNA as backups</span>'''
 +
 
 +
'''<span lang="EN-US">3)
 +
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>'''
 +
 
 +
''<span lang="EN-US">Performed as the usual protocol</span>''
 +
 
 +
<span lang="EN-US">1. Add 5ul DNA into 50ul competent cell. [Save the remaining 5ul DNA as backups]</span>
 +
 
 +
<span lang="EN-US">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>
 +
 
 +
<span lang="EN-US">3. Recover step: 200rpm, 45min, 18:44pm~19:30pm</span>
 +
 
 +
<span lang="EN-US">4. Centrifuge before distributing on agar plate: 4000rpm, 5min</span>
 +
 
 +
<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>
 +
 
 +
'''<span lang="EN-US">Results</span>：'''
 +
 
 +
'''<span lang="EN-US">Digested groups:</span>'''
 +
 
 +
<span lang="EN-US">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.</span>
 +
 
 +
'''<span lang="EN-US">Undigested groups:</span>'''
 +
 
 +
<span lang="EN-US">Some
 +
groups have colonies grown out on their undigested plates which can roughly
 +
indicate that our ligation was operative. </span>
-
Anyway, we still transferred DNA in the collecting tubes into new EP tubes, and then detect the concentration of them.
+
<span lang="EN-US"> </span>
-
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.
+
'''<span lang="EN-US">Improvement:</span>'''
-
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?
+
<span lang="EN-US">1) Increase
-
8. Enzyme Digestion for recovered DNA, to relinearize those sticky-end self-ligations
+
the quantity of DNA in each system </span>
-
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:
+
<span lang="EN-US">2) Heat
-
1) Increase the quantity of DNA in each system
+
inactivation before adding T4 DNA polymerase to avoid interplays.</span>
-
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.
+
 +
<span lang="EN-US">3)
 +
Perform restriction digestion directly after ligation step without DNA
 +
extraction.</span>
-
{{SUSTC-Shenzhen/main-content-end}}
+
<span lang="EN-US">4) If we failed again for the third time,
-
{{SUSTC-Shenzhen/wiki-footer}}
+
we plan to apply new method to eliminate the EcoRI site on UAS sequence. Such
-
{{SUSTC-Shenzhen/themeJs}}
+
as, site-specific mutagenesis, or isocaudarner ligation.</span>[[Category:VisualEditor]]*

Revision as of 10:03, 8 October 2014

Materials

The same as the first Fill-in experiment

Methods:

1. 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)

2. 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:

 

 

 

 

 

 

 

 

 

 

 

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.

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

1. Heat-inactivation 75°C，10min

2. Chill on ice, 15min

4. 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.

5. 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

6. 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

7. 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?

8. 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.*

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