Team:SUSTC-Shenzhen/Notebook/CRISPR/5*UAS-plasmids-Fill-in

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       goal=To eliminate the EcoRI site on the 5*UAS plasmid}}
       goal=To eliminate the EcoRI site on the 5*UAS plasmid}}
-
'''Materials:'''
+
==Materials==
-
 
+
* dNTPs
-
dNTPs
+
* Enzymes&Buffer:
-
 
+
* EcoRI, Cutsmart Buffer
-
Enzymes&Buffer:
+
* T4 DNA polymerase, T4 DNA polymerase Buffer
-
 
+
* T4 DNA ligase, T4 DNA ligase buffer
-
EcoRI, Cutsmart
+
* Kit:
-
Buffer
+
* TIANgel Purification Kit
-
 
+
==Methods==
-
T4 DNA polymerase,
+
===Restriction digestion for 5*UAS plasmid with EcoRI===
-
T4 DNA polymerase Buffer
+
{| class="table"
-
 
+
|-
-
T4 DNA ligase,
+
! Total volume !! Buffer !! DNA !! EcoRI !! ddH2O
-
T4 DNA ligase buffer
+
|-
-
 
+
| 20μl || 2μl || 8μl || 1μl || 9μl  
-
Kit:
+
-
 
+
-
  TIANgel
+
-
Purification Kit
+
-
 
+
-
'''Methods:'''
+
-
 
+
-
'''1. Restriction digestion
+
-
for 5*UAS plasmid with EcoRI'''
+
-
 
+
-
{|
+
-
+
-
  |
+
-
Total
+
-
  volume
+
-
 
+
-
  |
+
-
Buffer
+
-
 
+
-
  |
+
-
DNA
+
-
 
+
-
  |
+
-
EcoRI
+
-
 
+
-
  |
+
-
ddH<sub>2</sub>O 
+
-
+
-
|-
+
-
  |
+
-
20μl
+
-
 
+
-
  |
+
-
2μl
+
-
 
+
-
  |
+
-
8μl
+
-
 
+
-
  |
+
-
1μl
+
-
 
+
-
  |
+
-
9μl
+
-
+
|}
|}
-
 
Start time: 9:41am
Start time: 9:41am
-
 
End time: 2:00pm
End time: 2:00pm
-
 
+
===Electrophoresis & Agrose gel DNA extraction to remove enzymes and competent of Buffer===
-
'''2. Electrophoresis & Agrose
+
Loading system：
-
gel DNA extraction to remove enzymes and competent of Buffer'''
+
{| class="table"
-
 
+
|-
-
'''Loading system：'''
+
! Total volume/well !! DNA !! Dye !! TAE  
-
 
+
|-
-
{|
+
| 24μl || 5μl || 4μl || 15μl  
-
+
-
  |
+
-
Total volume/well
+
-
 
+
-
  |
+
-
DNA
+
-
 
+
-
  |
+
-
Dye
+
-
 
+
-
  |
+
-
TAE
+
-
+
-
|-
+
-
  |
+
-
24μl
+
-
 
+
-
  |
+
-
5μl
+
-
 
+
-
  |
+
-
4μl
+
-
 
+
-
  |
+
-
15μl
+
-
+
|}
|}
-
 
Running conditions：110V ， 30mim
Running conditions：110V ， 30mim
 +
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: 24.8ng/ul
 +
  G-PB5: 42.2ng/ul
 +
===Do 5’-end fill-in with T4 DNA polymerase===
 +
'''Protocol'''
-
'''DNA extraction:'''
+
# DNA should be dissolved in 1X NEBuffer 1-4 or T4 DNA Ligase Reaction Buffer supplemented with 100 µM each dNTP.
 +
# Add 1 unit T4 DNA Polymerase per microgram DNA and incubate 15 minutes at 12°C.
 +
# Stop reaction by adding EDTA to a final concentration of 10 mM and heating to 75°C for 20 minutes .
 +
#: CAUTION: Elevated temperatures, excessive amounts of enzyme, failure to supplement with dNTPs or long reaction times will result in recessed ends due to the 3´→ 5´ exonuclease activity of the enzyme.
 +
'''Reaction system:'''
-
  Performed
+
{| class="table"
-
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).
+
! Poly A
-
 
+
! G-PB5
-
'''Concentration
+
|-
-
of the purified DNA fragments:'''
+
| DNA
-
 
+
| 17.8ul(441.44ng)
-
  
+
| 17.8ul(751.16ng)
-
Poly A: 24.8ng/ul
+
|-
-
 
+
| 10X T4 DNA Ligase Reaction Buffer
-
  
+
| 2ul
-
G-PB5: 42.2ng/ul
+
| 2ul
-
 
+
|-
-
'''3. Do 5’-end fill-in with T4
+
| dNTP
-
DNA polymerase'''
+
| 0.2ul
-
 
+
| 0.2ul
-
'''1）Protocol：'''
+
|-
-
 
+
| T4 polymerase (3U/ul)
-
1）DNA should be dissolved
+
| 0.2ul (Theoretical value: 0.1471ul)
-
in 1X NEBuffer 1-4 or T4 DNA Ligase Reaction Buffer supplemented with 100 µM
+
| 0.3ul (Theoretical value: 0.2503ul)
-
each dNTP.
+
|-
-
 
+
| Total volume
-
2）Add 1 unit T4 DNA
+
| 20.3ul
-
Polymerase per microgram DNA and incubate 15 minutes at 12°C.
+
| 20.2ul
-
 
+
-
3）Stop reaction by
+
-
adding EDTA to a final concentration of 10 mM and heating to 75°C for 20
+
-
minutes .
+
-
 
+
-
CAUTION: Elevated temperatures, excessive
+
-
amounts of enzyme, failure to supplement with dNTPs or long reaction times will
+
-
result in recessed ends due to the 3´→ 5´ exonuclease
+
-
activity of the enzyme.
+
-
 
+
-
'''2）Reaction
+
-
system：'''
+
-
 
+
-
{|
+
-
+
-
  |
+
-
''' '''
+
-
 
+
-
  |
+
-
'''Poly
+
-
  A'''
+
-
 
+
-
  |
+
-
'''G-PB5''' 
+
-
+
-
|-
+
-
  |
+
-
'''DNA'''
+
-
 
+
-
  |
+
-
'''17.8ul'''(441.44ng)
+
-
 
+
-
  |
+
-
'''17.8ul'''(751.16ng)
+
-
+
-
|-
+
-
  |
+
-
'''10X
+
-
  T4 DNA Ligase Reaction Buffer'''
+
-
 
+
-
  |
+
-
'''2ul'''
+
-
 
+
-
  |
+
-
'''2ul''' 
+
-
+
-
|-
+
-
  |
+
-
'''dNTP'''
+
-
 
+
-
  |
+
-
'''0.2ul'''
+
-
 
+
-
  |
+
-
'''0.2ul''' 
+
-
+
-
|-
+
-
  |
+
-
'''T4
+
-
  polymerase'''
+
-
 
+
-
'''(3U/ul)'''
+
-
 
+
-
  |
+
-
'''0.2ul'''
+
-
 
+
-
'''(Theoretical
+
-
  value: 0.1471ul)'''
+
-
 
+
-
  |
+
-
'''0.3ul'''
+
-
 
+
-
'''(Theoretical
+
-
  value: 0.2503ul)''' 
+
-
+
-
|-
+
-
  |
+
-
'''Total
+
-
  volume'''
+
-
 
+
-
  |
+
-
'''20.3ul'''
+
-
 
+
-
  |
+
-
'''20.2ul''' 
+
-
+
|}
|}
-
'''3) EDTA: we prepared 100mM EDTA stock solution.'''
+
'''EDTA'''
-
To stop the
+
We prepared 100mM EDTA stock solution.
-
reaction, we added 1ul EDTA (100mM) to 19ul reaction liquid. Heat 75°C, 20min
+
To stop the reaction, we added 1ul EDTA (100mM) to 19ul reaction liquid. Heat 75°C, 20min
 +
'''Blunt end ligation with T4 DNA ligase'''
-
'''4. Blunt end ligation with
+
# Till now, the concentration of DNA  
-
T4 DNA ligase'''
+
#: Poly A reaction system：21.0ng/ul
-
 
+
#: G-PB5 reaction system：35.3ng/ul
-
'''1)'''
+
# ligation system：
-
'''Till now, the concentration of DNA '''
+
#:
-
 
+
{| class="table"
-
Poly A reaction system：21.0ng/ul
+
!
-
 
+
! DNA
-
        G-PB5 reaction system：35.3ng/ul
+
! 10X buffer
-
 
+
! T4 ligase
-
'''2) ligation system'''：
+
! ddH2O
-
 
+
|-
-
{|
+
| Theoretical volume
-
+
| 37.5ng
-
  |
+
| 2ul
-
 
+
| 1ul
-
 
+
| Add to 20ul
-
  |
+
|-
-
DNA
+
| Poly A(1,2)
-
 
+
| 2ul
-
  |
+
| 2ul
-
10X
+
| 1ul
-
  buffer
+
| 15ul
-
 
+
|-
-
  |
+
| G-PB5(1,2)
-
T4
+
| 1ul
-
  ligase
+
| 2ul
-
 
+
| 1ul
-
  |
+
| 16ul
-
ddH<sub>2</sub>O 
+
-
+
-
|-
+
-
  |
+
-
Theoretical
+
-
  volume
+
-
 
+
-
  |
+
-
37.5ng
+
-
 
+
-
  |
+
-
2ul
+
-
 
+
-
  |
+
-
1ul
+
-
 
+
-
  |
+
-
Add
+
-
  to 20ul
+
-
+
-
|-
+
-
  |
+
-
Poly
+
-
  A(1,2)
+
-
 
+
-
  |
+
-
2ul
+
-
 
+
-
  |
+
-
2ul
+
-
 
+
-
  |
+
-
1ul
+
-
 
+
-
  |
+
-
15ul
+
-
+
-
|-
+
-
  |
+
-
G-PB5(1,2)
+
-
 
+
-
 
+
-
  |
+
-
1ul
+
-
 
+
-
  |
+
-
2ul
+
-
 
+
-
  |
+
-
1ul
+
-
 
+
-
  |
+
-
16ul
+
-
+
|}
|}
-
'''2) Protocol: '''
+
# Protocol:  
-
 
+
## Incubate 16°C, overnight (16h)
-
  1.
+
## Heat inactivation, 65°C, 10min
-
Incubate 16°C, overnight (16h)
+
## Chill on ice
-
 
+
# Remove EDTA:  
-
  2. Heat inactivation, 65°C, 10min
+
#: we add 1ul pfu (with MgSO4) buffer to each ligation system (totally, 4*tubes: polyA1, polyA2, G1, G2), in order to neutralize the inhibit effect of EDTA.
-
 
+
# Restriction digestion using EcoRI to re-linearize those sticky end ligations, thus selecting out those successful ones.
-
  3. Chill on ice
+
## Till now, the concentration of DNA  
-
 
+
##: Poly A reaction system：37.5ng/20ul=1.8ng/ul
-
'''3)
+
##: G-PB5 reaction system：37.5ng/20ul=1.8ng/ul
-
Remove EDTA:'''
+
## Digestion system:
-
 
+
##:
-
we add 1ul ''pfu'' (with MgSO<sub>4</sub>) buffer to each ligation system
+
{| class="table"
-
[totally, 4*tubes: polyA1, polyA2, G1, G2], in order to neutralize the inhibit
+
!
-
effect of EDTA.
+
! Total volume
-
 
+
! EcoRI (20U/ul)
-
'''5. Restriction digestion
+
! DNA
-
using EcoRI to re-linearize those sticky end ligations, thus selecting out
+
! 10X NEBuffer
-
those successful ones.'''
+
! ddH2O
-
 
+
|-
-
'''1)'''
+
| Theoretical volume
-
'''Till now, the concentration of DNA '''
+
| 10ul
-
 
+
| 1 unit
-
Poly A reaction system：37.5ng/20ul=1.8ng/ul
+
| X ul (0.1ug)
-
 
+
| 1ul
-
G-PB5 reaction system：37.5ng/20ul=1.8ng/ul
+
| Add to 10ul
-
 
+
|-
-
'''2)
+
| Poly A1 & G-PB51
-
Digestion system:'''
+
| 22.1ul
-
 
+
| 0.1ul (2 unit, obviously we have excessive enzyme)
-
{|
+
| 20ul(about 37.5ng)
-
+
| 2ul
-
  |
+
| -
-
 
+
-
 
+
-
  |
+
-
Total
+
-
  volume
+
-
 
+
-
  |
+
-
EcoRI
+
-
  (20U/ul)
+
-
 
+
-
  |
+
-
DNA
+
-
 
+
-
  |
+
-
10X
+
-
  NEBuffer
+
-
 
+
-
  |
+
-
ddH<sub>2</sub>O 
+
-
+
-
|-
+
-
  |
+
-
Theoretical volume
+
-
 
+
-
  |
+
-
10ul
+
-
 
+
-
  |
+
-
1
+
-
  unit
+
-
 
+
-
  |
+
-
X
+
-
  ul  
+
-
 
+
-
(0.1ug)
+
-
 
+
-
  |
+
-
1ul
+
-
 
+
-
  |
+
-
Add
+
-
  to 10ul
+
-
+
-
|-
+
-
  |
+
-
Poly A1 &
+
-
  G-PB51
+
-
 
+
-
  |
+
-
22.1ul
+
-
 
+
-
  |
+
-
0.1ul
+
-
  (2
+
-
  unit, 显然酶过量了)
+
-
 
+
-
  |
+
-
20ul(大约37.5ng)
+
-
 
+
-
  |
+
-
2ul
+
-
 
+
-
  |
+
-
<nowiki>-</nowiki> 
+
-
+
|}
|}
-
'''Also, design a
+
''Also, design a non-digestion system as control''
-
non-digestion system as control'''
+
-
Poly A2 &
+
Poly A2 & G-PB52 didn’t undergo restriction enzyme digestion.  
-
G-PB52 didn’t undergo restriction enzyme digestion.  
+
-
'''6. Transformation '''
+
'''Transformation'''
''Performed as the usual protocol''
''Performed as the usual protocol''
 +
# 5ul ligation reaction to 50ul competent cell.
 +
# Incubate: 2014.8.7 9:30pm~2014.8.8 2:30pm (17h)
-
5ul ligation
+
==Results==
-
reaction to 50ul competent cell.
+
-
 
+
-
Incubate: 2014.8.7
+
-
9:30pm~2014.8.8 2:30pm (17h)
+
-
 
+
-
'''Results:'''
+
-
 
+
-
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+
-
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+
-
 
+
-
1) From the
+
-
picture, we can see that, there were no colonies growing on the plate that were
+
-
grown with plasmid-digested bacteria（Poly A1, G1）. While for those bacteria which were
+
-
transformed with undigested plasmid (the control group: Poly A2,
+
-
G2),
+
-
several colonies have already appeared on the agar plate.
+
-
 
+
-
2) This meant the successfully transformed colonies were all false
+
-
positive, of which the plasmids were all formed by sticky-end self-ligation,
+
-
rather than blunt ends which should be generated via 5’ end Fill-in.
+
-
 
+
-
3) Finally, in a word, our 5*UAS plasmids Fill-in has failed.
+
-
 
+
-
'''Points that can be improved
+
-
in the next time：'''
+
-
 
+
-
1. Do not undergo
+
-
Agrose gel DNA extraction after the-first-time enzyme digestion, since the
+
-
buffer for digestion is also suitable to T4 polymerase. Thus, a relatively high
+
-
concentration of DNA can be assured.
+
-
2. After the
+
{{SUSTC-Image|wiki/images/1/10/SUSTC-Image-5UAS-Fillin-1.jpg}}
-
polyreaction step of Fill-in, recover DNA by agarose gel DNA extraction, to
+
# From the picture, we can see that, there were no colonies growing on the plate that were grown with plasmid-digested bacteria（Poly A1, G1）. While for those bacteria which were transformed with undigested plasmid (the control group: Poly A2, G2), several colonies have already appeared on the agar plate.  
-
remove EDTA,
+
# This meant the successfully transformed colonies were all false positive, of which the plasmids were all formed by sticky-end self-ligation, rather than blunt ends which should be generated via 5’ end Fill-in.
-
buffer etc. most of which might affect the following experiment.
+
# Finally, in a word, our 5*UAS plasmids Fill-in has failed.
-
3. Design a concentration gradient when performing the T4 ligation step
+
Points that can be improved in the next time：
-
(30ng, 100ng, 200ng DNA). Make sure that there is always a DNA concentration
+
# Do not undergo Agrose gel DNA extraction after the-first-time enzyme digestion, since the buffer for digestion is also suitable to T4 polymerase. Thus, a relatively high concentration of DNA can be assured.
-
involved in the optimum range for ligation.
+
# After the polyreaction step of Fill-in, recover DNA by agarose gel DNA extraction, to remove EDTA, buffer etc. most of which might affect the following experiment.
 +
# Design a concentration gradient when performing the T4 ligation step (30ng, 100ng, 200ng DNA). Make sure that there is always a DNA concentration involved in the optimum range for ligation.

Latest revision as of 13:40, 17 October 2014

Team SUSTC-Shenzhen

Notebook

Elements of the endeavor.

5*UAS plasmids Fill-in

2014/8/6 To eliminate the EcoRI site on the 5*UAS plasmid

Materials

  • dNTPs
  • Enzymes&Buffer:
  • EcoRI, Cutsmart Buffer
  • T4 DNA polymerase, T4 DNA polymerase Buffer
  • T4 DNA ligase, T4 DNA ligase buffer
  • Kit:
  • TIANgel Purification Kit

Methods

Restriction digestion for 5*UAS plasmid with EcoRI

Total volume Buffer DNA EcoRI ddH2O
20μl 2μl 8μl 1μl 9μl

Start time: 9:41am End time: 2:00pm

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

Loading system：

Total volume/well DNA Dye TAE
24μl 5μl 4μl 15μl

Running conditions：110V ， 30mim 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: 24.8ng/ul
  G-PB5: 42.2ng/ul

Do 5’-end fill-in with T4 DNA polymerase

Protocol

  1. DNA should be dissolved in 1X NEBuffer 1-4 or T4 DNA Ligase Reaction Buffer supplemented with 100 µM each dNTP.
  2. Add 1 unit T4 DNA Polymerase per microgram DNA and incubate 15 minutes at 12°C.
  3. Stop reaction by adding EDTA to a final concentration of 10 mM and heating to 75°C for 20 minutes .
    CAUTION: Elevated temperatures, excessive amounts of enzyme, failure to supplement with dNTPs or long reaction times will result in recessed ends due to the 3´→ 5´ exonuclease activity of the enzyme.

Reaction system:

Poly A G-PB5
DNA 17.8ul(441.44ng) 17.8ul(751.16ng)
10X T4 DNA Ligase Reaction Buffer 2ul 2ul
dNTP 0.2ul 0.2ul
T4 polymerase (3U/ul) 0.2ul (Theoretical value: 0.1471ul) 0.3ul (Theoretical value: 0.2503ul)
Total volume 20.3ul 20.2ul

EDTA

We prepared 100mM EDTA stock solution. To stop the reaction, we added 1ul EDTA (100mM) to 19ul reaction liquid. Heat 75°C, 20min Blunt end ligation with T4 DNA ligase

  1. Till now, the concentration of DNA
    Poly A reaction system：21.0ng/ul
    G-PB5 reaction system：35.3ng/ul
  2. ligation system：
DNA 10X buffer T4 ligase ddH2O
Theoretical volume 37.5ng 2ul 1ul Add to 20ul
Poly A(1,2) 2ul 2ul 1ul 15ul
G-PB5(1,2) 1ul 2ul 1ul 16ul
  1. Protocol:
    1. Incubate 16°C, overnight (16h)
    2. Heat inactivation, 65°C, 10min
    3. Chill on ice
  2. Remove EDTA:
    we add 1ul pfu (with MgSO4) buffer to each ligation system (totally, 4*tubes: polyA1, polyA2, G1, G2), in order to neutralize the inhibit effect of EDTA.
  3. Restriction digestion using EcoRI to re-linearize those sticky end ligations, thus selecting out those successful ones.
    1. Till now, the concentration of DNA
      Poly A reaction system：37.5ng/20ul=1.8ng/ul
      G-PB5 reaction system：37.5ng/20ul=1.8ng/ul
    2. Digestion system:
Total volume EcoRI (20U/ul) DNA 10X NEBuffer ddH2O
Theoretical volume 10ul 1 unit X ul (0.1ug) 1ul Add to 10ul
Poly A1 & G-PB51 22.1ul 0.1ul (2 unit, obviously we have excessive enzyme) 20ul(about 37.5ng) 2ul -

Also, design a non-digestion system as control

Poly A2 & G-PB52 didn’t undergo restriction enzyme digestion.

Transformation

Performed as the usual protocol

  1. 5ul ligation reaction to 50ul competent cell.
  2. Incubate: 2014.8.7 9:30pm~2014.8.8 2:30pm (17h)

Results

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  1. From the picture, we can see that, there were no colonies growing on the plate that were grown with plasmid-digested bacteria（Poly A1, G1）. While for those bacteria which were transformed with undigested plasmid (the control group: Poly A2, G2), several colonies have already appeared on the agar plate.
  2. This meant the successfully transformed colonies were all false positive, of which the plasmids were all formed by sticky-end self-ligation, rather than blunt ends which should be generated via 5’ end Fill-in.
  3. Finally, in a word, our 5*UAS plasmids Fill-in has failed.

Points that can be improved in the next time：

  1. Do not undergo Agrose gel DNA extraction after the-first-time enzyme digestion, since the buffer for digestion is also suitable to T4 polymerase. Thus, a relatively high concentration of DNA can be assured.
  2. After the polyreaction step of Fill-in, recover DNA by agarose gel DNA extraction, to remove EDTA, buffer etc. most of which might affect the following experiment.
  3. Design a concentration gradient when performing the T4 ligation step (30ng, 100ng, 200ng DNA). Make sure that there is always a DNA concentration involved in the optimum range for ligation.


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Licensed under CC BY 4.0.

  • ©2014 SUSTC-Shenzhen

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