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=}}
==Materials==
==Materials==
-
The same as the first Fill-in experiment
+
 
 +
The same as the first Fill-in experiment.
==Methods==
==Methods==
-
1. Restriction digestion for 5*UAS plasmid with EcoRI（unit: ul）
+
 
-
{| class="table"
+
===Restriction digestion for 5*UAS plasmid with EcoRI (unit: ul)===
-
|-
+
 
-
!  !! Total volume !! 10X Buffer !! DNA !! EcoRI !! ddH2O  
+
{|class="table"
 +
!
 +
!Total volume
 +
!10X Buffer
 +
!DNA
 +
!EcoRI
 +
!ddH2O
|-
|-
-
| Poly A(168.8ng/ul) || 20 || 2 || 2.5 || 1 || 14.5  
+
|Poly A(168.8ng/ul)
 +
|20
 +
|2
 +
|2.5
 +
|1
 +
|14.5
|-
|-
-
| G-PB5(293.6ng/ul) || 20 || 2 || 2 || 1 || 15  
+
|G-PB5(293.6ng/ul)
 +
|20
 +
|2
 +
|2
 +
|1
 +
|15  
|}
|}
-
Time: 2014.8.8 10:00pm ~ 2014.8.9 9:30am ( Incubate overnight)
+
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
+
===Electrophoresis to verify if most of the digested plasmid has been linearized===
-
Loading system：（unit: ul）
+
 
-
{| class="table"
+
'''Loading system：'''(unit: ul)
-
|-
+
{|class="table"
-
! Total volume/well !! DNA !! Dye !! TAE  
+
|Total volume/well
 +
|DNA
 +
|Dye
 +
|TAE
|-
|-
-
| 12 || 0.5 || 2 || 9.5  
+
|12
 +
|0.5
 +
|2
 +
|9.5
|}
|}
-
Running conditions: 130V,   30min
+
Running conditions: 130V,   30min
-
Results of electrophoresis:
+
 +
'''Results of electrophoresis:'''
 +
{{SUSTC-Image|wiki/images/6/63/SUSTC-Shenzhen-UAS-2nd-1.PNG}}
 +
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)'''
 +
{|class="table"
 +
|
 +
|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:'''
-
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
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.
+
b. add 2ul EDTA(100mM) to 18ul reaction
-
4. Accident：
+
system to stop the polyreaction.  
-
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
+
c. put in 65°C，20min to
-
DNA extraction:
+
inactivate T4 DNA polymerase thoroughly.
-
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:
+
'''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
Poly A: 19.3ng/ul
 +
G-PB5: 18.8ng/ul
G-PB5: 18.8ng/ul
-
6. Blunt end ligation with T4 DNA ligase [Design a concentration gradient]
+
 
-
1) Ligation system：（unit: ul）
+
===Blunt end ligation with T4 DNA ligase [Design a concentration gradient]===
-
Total volume DNA 10X T4 DNA ligase buffer T4 ligase EcoRI
+
 
-
(to reduce those sticky ends ligation) ddH2O
+
'''1) Ligation system：'''（unit: ul）
-
(40ng rank)
+
{|class="table"
-
1.2 tube 21 2 2 1 1 15
+
|
-
(100ng rank)
+
|Total volume
-
3,4 tube 21 6 2 2 1 10
+
|DNA
-
(200ng rank)
+
|10X T4 DNA ligase buffer
-
5,6 tube 21 12 2 2 1 4
+
|T4 ligase
-
2) Protocol：
+
|EcoRI (to reduce those sticky ends ligation)
-
a. incubate the ligation system at 16°C，15h [8.9 1:30pm ~ 8.10 4:30am ]
+
|ddH2O
-
b. store at 4°C [8.10 4:30am ~ 10:00am]
+
|-
 +
|(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
c. Heat inactivation, 65°C, 10min
 +
d. chill on ice
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.
+
===Electrophoresis & Agrose gel DNA extraction to remove enzymes and competent of Buffer===
-
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.
+
'''DNA extraction:'''
-
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
+
 +
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.
 +
{|class="table"
 +
|
 +
|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.
 +
{|class="table"
 +
|
 +
|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?
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]
+
===Enzyme Digestion for recovered DNA, to relinearize those sticky-end self-ligations===
-
Total volume DNA EcoRI 10X NEBuffer
+
 
-
10 8.5 0.5 1
+
'''1) Digestion system''' [unit: ul]
-
2) Reserve the rest 10ul DNA as backups
+
{|class="table"
-
3) incubate the digestion system 37°C， 4h [12:35am ~ 5:00am]
+
|Total volume
-
9. Transformation
+
|DNA
-
Performed as the usual protocol
+
|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]
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.
+
 
 +
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
3. Recover step: 200rpm, 45min, 18:44pm~19:30pm
 +
4. Centrifuge before distributing on agar plate: 4000rpm, 5min
4. Centrifuge before distributing on agar plate: 4000rpm, 5min
 +
5. Incubate, 37°C, overnight [2014.8.10 8:44pm~2014.8.11 10:00am]
5. Incubate, 37°C, overnight [2014.8.10 8:44pm~2014.8.11 10:00am]
-
Results：
+
 
-
Digested groups:
+
==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.
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:
+
===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  
1) Increase the quantity of DNA in each system  
 +
2) Heat inactivation before adding T4 DNA polymerase to avoid interplays.
2) Heat inactivation before adding T4 DNA polymerase to avoid interplays.
 +
3) Perform restriction digestion directly after ligation step without DNA extraction.
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.
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.

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.


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