Team:NYMU-Taipei/Experiments/Riboswitch

From 2010.igem.org

(Difference between revisions)
(β†’2010.10.03)
(β†’2010.10.22)
 
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|1: marker 100bp|||n|=
|1: marker 100bp|||n|=
|2: -|-|-|n|=
|2: -|-|-|n|=
-
|3: riboswitch |56 bp|none|w|=
+
|3: riboswitch |56 bp|none|f|=
-
|4: riboswitch |56 bp|none|w|=
+
|4: riboswitch |56 bp|none|f|=
|5: -|-|-|n|=
|5: -|-|-|n|=
-
|6: riboswitch |56 bp|none|w|=
+
|6: riboswitch |56 bp|none|f|=
-
|7: riboswitch |56 bp|none|w|=
+
|7: riboswitch |56 bp|none|f|=
|8: -|-|-|n|=
|8: -|-|-|n|=
-
|9: riboswitch |56 bp|none|w|=
+
|9: riboswitch |56 bp|none|f|=
-
|10: riboswitch |56 bp|none|w|=
+
|10: riboswitch |56 bp|none|f|=
-
|11: Positive Control|1100 bp|none|f|=
+
|11: Positive Control|1100 bp|none|w|=
-
|12: Negative Control||Contamination ~300bp |f|=
+
|12: Negative Control||Contamination ~300bp |w|=
|}}  
|}}  
{| border=1
{| border=1
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=2010.08.23=
=2010.08.23=
-
*the upper experiment are failed,so we restart the experiment.
+
*the upper experiment are failed, so we restart the experiment.
**Run gel 11:00
**Run gel 11:00
**After nanodrop, we found that the concentration is too low(only 3.0), so we re-PCR-of-primer again.
**After nanodrop, we found that the concentration is too low(only 3.0), so we re-PCR-of-primer again.
Line 1,027: Line 1,027:
{| border="1"
{| border="1"
|-
|-
-
! Total: !! 49μl || X1.7μl ||   
+
! Total: !! 49μl || X1.7  ||   
|-
|-
| FP || 1μl || 1.7μl || Theophylline Riboswitch Forward Primer     
| FP || 1μl || 1.7μl || Theophylline Riboswitch Forward Primer     
Line 1,081: Line 1,081:
{| border="1"
{| border="1"
|-
|-
-
! Total: !! 49μl || X1.9μl 
+
! Total: !! 49μl || X1.9
|-
|-
| VR+VF|| 2μl || 3.8μl  
| VR+VF|| 2μl || 3.8μl  
Line 1,130: Line 1,130:
{| border="1"
{| border="1"
|-
|-
-
! Total: !! 49μl || X2.5μl   
+
! Total: !! 49μl || X2.5  
|-
|-
| VR+VF|| 2μl || 5μl   
| VR+VF|| 2μl || 5μl   
Line 1,138: Line 1,138:
| 10XBuff. || 5μl || 12.5μl  
| 10XBuff. || 5μl || 12.5μl  
|-
|-
-
| tag || 0.25μl || 1.25mu;l   
+
| tag || 0.25μl || 1.25μl   
|-
|-
| ddH2O ||  39.75μl || 99.375μl
| ddH2O ||  39.75μl || 99.375μl
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{| border="1"
{| border="1"
|-
|-
-
! Total: !! 49μl || X1.6μl  
+
! Total: !! 49μl || X1.6   
|-
|-
| VR+VF|| 2μl || 3.2μl   
| VR+VF|| 2μl || 3.2μl   
Line 1,205: Line 1,205:
| 10XBuff. || 5μl || 8μl   
| 10XBuff. || 5μl || 8μl   
|-
|-
-
| tag || 0.25μl || 0.4mu;l   
+
| tag || 0.25μl || 0.4μl   
|-
|-
| ddH2O ||  39.75μl || 63.6μl   
| ddH2O ||  39.75μl || 63.6μl   
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*Transform
*Transform
-------------------------------
-------------------------------
-
*Theophylline solution; concentration is 0.1M
+
*Preparing 0.1 M Theophylline solution in DMSO.
*Assay
*Assay
{| border=1
{| border=1

Latest revision as of 23:56, 27 October 2010


Contents

Parts

2010.08.17

  • PCR of primer&Digestion&Ligation
NYMU Ribo.jpg

Descr Win length Fail length
1: marker 100bp
2: - - -
3: riboswitch 56 bp none
4: riboswitch 56 bp none
5: - - -
6: riboswitch 56 bp none
7: riboswitch 56 bp none
8: - - -
9: riboswitch 56 bp none
10: riboswitch 56 bp none
11: Positive Control 1100 bp none
12: Negative Control Contamination ~300bp
PCR mixfor 3 tubes(25+25+50) total 50 μl temp time
template 1μl 94oC 120s
forward primer 1μ1 94oC 30s
reverse primer 1μ1 55oC 30s
dNTP 4μ1 72oC 40s
10x buffer 5μl 72oC300s
ddH2O 39.75μl
pfu 0.5μ1
  • Plasmid extraction
  • Digestion
  • PCR purification(centrifuge)
  • Nanodrop
  • Ligation
  • Transform 20:00

2010.08.18

  • PCR mix
  • Run gel(2%argarose 100v)
NYMU CIMG1185.jpg

Descr Win length Fail length
1: marker 100bp
2: th-ribo gene + vector 294bp -
3: th-ribo gene + vector 294bp -
4: - - -
5: Positive Control 1100 bp none
6: Negative Control Contamination ~3000bp
Total:(*2) 50μl
template 2μl
VF+VR 2μl
dNTP 2μl
Buffer 5μl
ddH2O 39.75μl
Enzyme tag 0.25μl

(negative contaminated and appeared three different bands)

  • 3-in-1 11:30
  • Run PCR 12:00
  • put Liquid culture&plate at 17:00

2010.08.19

  • Because there are some mistake resulting from gel running, we decided to digest again.
    • Digest 10:30
    • Transformation 12:45
  • At the same time, we continued plasmid extraction of the result one of yesterday.
    • plasmid extraction 10:30
    • Digest 11:30
    • run gel 13:00
    • Ligation 14:00
    • Transformation 14:30

2010.08.20

  • We decide to re-3-in-1 again.
  • PCR mix 10:30
  • 3 in 1 11:30
  • Run PCR 01:00~02:30
  • Run gel 15:00(100v 20 mins)
NYMU R20.JPG

Descr Win length Fail length
1: marker 100bp
2: ribo+vector 294bp
3: ribo+vector 294bp
4: - - -
5: Positive Control 1100 bp none
6: Negative Control
Total:(*2) 50μl
template 2μl
VF+VR 2μl
dNTP 2μl
Buffer 5μl
ddH2O 39.75μl
Enzyme tag 0.25μl
  • Cut gel 16:30
  • Plasmid extraction 16:30

2010.08.23

  • the upper experiment are failed, so we restart the experiment.
    • Run gel 11:00
    • After nanodrop, we found that the concentration is too low(only 3.0), so we re-PCR-of-primer again.
  • PCR of primer 16:00
  • Transformation(GFP) 17:15

2010.08.24

  • Run gel 10:30am(5ul)
NYMU R24.JPG

Descr Win length Fail length
1:
2: marker 100bp
3:
4: pcr riboswitch 56bp
5:
6: Positive Control 300bp
7: Negative Control
Total:(*2) 50μl
template 2μl
VF+VR 2μl
dNTP 2μl
Buffer 5μl
ddH2O 39.75μl
Enzyme tag 0.25μl
  • PCR Purify 10:45(45ul)
  • Digest 11:15
Total: 30μl
DNA 26μl
Digestion buffer 2μl
Enzyme 1(X) 1μl
Enzyme 2(P) 1μl
  • Digest purify 13:30
  • Nanodrop(19.8ng/ul)
  • Ligation 15:44
    • Ligate riboswitch+pSB1A2(1:3) and ligate GFP+pSB1A2(1:2)(we use 2009's GFP+terminater so we have to ligate pSB1A2)
Total: 10μl
DNA of vector 1μl
DNA of insert 3μl
buffer 2μl
ligase 0.5μl
ddH2O 4.5μl
Total: 10μl
DNA of vector 1μl
DNA of insert 2μl
buffer 2μl
ligase 0.5μl
ddH2O 5.5μl

2010.08.25

  • 3-in-1:GFP(1 colony),Riboswitch(2 colony) 17:00pm
  • Run PCR 17:20pm

2010.08.26

  • Run gel 10:30am
    • Because we thought the result is unsure,we decide to digest to check.
NYMU R26-2.JPG

Descr Win length Fail length
1: GFP 720bp
2: (1)riboswitch+pSB1A2 294bp
3: (2)riboswitch+pSB1A2 294bp
4: Positive Control 1100bp
5: Negative Control Contamination~1000 bp
6: Marker:100bp
Total:(*2) 50μl
template 2μl
VF+VR 2μl
dNTP 2μl
Buffer 5μl
ddH2O 39.25μl
Enzyme tag 0.25μl
  • Liquid culture 12:00
    • the first step when we added MX1 we didn't resuspend,so we readded MX1~MX3.But the DNA may be less.
  • digest
    • [GFP(XP),ribo1(SP,XP>>use 10 ul and 20ul protocal),ribo2(SP,XP>>use 10 ul and use 20ul protocal)]
Total: 10μl
DNA of vector(sp) 1μl
DNA of insert(xp) 3μl
buffer 2μl
ligase 0.5μl
ddH2O 4.5μl
  • run gel(xp)
NYMU R26.JPG

Descr Win length Fail length
1: ribo1 96bp
2:
3: ribo2 96bp
4:
5: Marker:100bp
6: GFP 854bp
Total:(*2) 50μl
template 2μl
VF+VR 2μl
dNTP 2μl
Buffer 5μl
ddH2O 39.75μl
Enzyme tag 0.25μl
  • purify(ribo-1(sp);ribo-2(sp);GFP(xp))
  • ligate
  • transform
  • liquid culture from the plate(8/24)

2010.08.27

  • PCR mix 10:30
    • GFP+terminator+Riboswitch=1157bp
  • 3 in 1 12:15
  • Run PCR 12:30~13:30
  • Run gel 13:45

  • Liquid culture again(use the plate 8/24) 10:30
    • To check the wrong step of liquid culture
  • Digestion 13:40
  • Run gel 01:45pm[GFP(XP).Riboswitch-1(XP).Riboswitch-2(XP)]
NYMU R27.JPG

Descr Win length Fail length
1:FRV 1158bp 294bp
2: FRV 1158bp 294bp
3: 100bp marker - -
4: FRV 1158bp 294bp
5: positive control 1100bp
6: Negative Control - Contamination ~300bp
Total:(*2) 50μl
template 2μl
VF+VR 2μl
dNTP 2μl
Buffer 5μl
ddH2O 39.75μl
Enzyme tag 0.25μl

2010.08.28

  • 3-IN-1 (another 3 colony from 08.26 Riboswitch+GFP-Terminator plate) 13:30
    • PCR (Use new dNTP & Taq buffer & taq) 14:30
    • Run Gel 16:20
NYMU P9010273.jpg

Descr Win length Fail length
1: FRV 1158bp 294bp
2: FRV 1158bp 294bp
3: FRV 1158bp 294bp
4: positive control 1100bp -
5: marker100 bp - -
6: negative control - -
Total:(*2) 50μl temptime
template 2μl 94oC60s
VF+VR 2μl94oC15s
dNTP 2μl55oC20s
Buffer 5μl72oC100s
ddH2O 39.75μl72oC300s
Enzyme tag 0.25μl
  • Transformation GFP+vector 17:00
  • 3-IN-1 (2 Colony from the plate transformed on 08/27) 17:20
    • PCR 18:25~19:40

2010.08.29

  • Run Gel (with green marker) at 11:00
    • Ribo Vac1 and Ribo Vac2 from PCR on 8/28 at 18:25~19:40
NYMU P9010274.jpg

Descr Win length Fail length
1: - - -
2: - - -
3: RV1 294bp 1100bp
4: marker100 bp - -
5: RV2 294bp -
6: positive control 1100bp -
7: negative control - -
Total:(*2) 50μl
template 2μl
VF+VR
dNTP 2μl
Buffer 5μl
ddH2O 39.75μl
Enzyme tag 0.25μl

______________________________________________________________________________________________________________

  • 3-in-1 (3 colonies of GFP+TERMINATOR and 1 of Riboswitch+PSB1A2 vector, both from plates transformed on 8/28)
    • PCR 11:00~12:50
    • Run Gel 15:30 (result shows no band!! but positive and negative controls are correct)
NYMU P9010276.jpg

Descr Win length Fail length
1: GFP1 854bp
2: GFP2 854bp
3: GFP3 854bp
4: marker100 bp - -
5: riboswitch+vector 294bp -
6: negative control - -
7: positive contril 1100bp -
Total:(*2) 50μl
template 2μl
VF+VR 2μl
dNTP 2μl
Buffer 5μl
ddH2O 39.75μl
Enzyme tag 0.25μl
  • transform 16:30
    • (1)the Ribo2 from 8.17 (2)the Ribo (check) from 08/19 (3)GFP+terminator from 08/27
      • put into incubator 17:30

2010.08.30

  • 3-in-1: GFP,GFP+riboswitch,ribo-1,ribo-2 12:30pm
  • Run PCR 13:20
  • Run gel(2% argarose 100v)
NYMU P9010277.jpg

Descr Win length Fail length
1: GFP1 854bp -
2: GFP2 854bp -
3: GFP3 854bp -
4: riboswitch1+GFP 1158bp -
5: riboswitch2+GFP 1158bp -
6: riboswitch3+GFP 1158bp -
7: marker100 bp - -
8: ribo1+vector 294bp -
9: ribo1+vector 294bp -
10: ribo2+vector 294bp -
11: ribo2+vector 294bp -
12: positive control 1100bp -
13: negative control - -
Total:(*2) 50μl
template 2μl
VF+VR
dNTP 2μl
Buffer 5μl
ddH2O 39.75μl
Enzyme tag 0.25μl
  • Finally we Transform 19F(GFP+terminator), E0040 18:50

2010.08.31

  • 3-in-1 (ribo(xp)+pSB1A2,E0040,J04630)11:00
  • Nanodrop
  • Ligation
NYMU P9010280.jpg

Descr Win length Fail length
1: GFP1(J04630) 854bp -
2: GFP2(J04630) 854bp -
3: Riboswitch1+vector 294bp -
4: Riboswitch+2vector 294bp -
5: marker 100 bp - -
6: GFP1(E0040) 720bp -
7: GFP2(E0040) 720bp -
8: ribo1+vector 294bp -
9: ribo1+vector 294bp -
10: ribo2+vector 294bp -
11: ribo2+vector 294bp -
12: positive control 1100bp -
13: negative control - -
Total:(*2) 50μl
template 2μl
VF+VR
dNTP 2μl
Buffer 5μl
ddH2O 39.75μl
Enzyme tag 0.25μl
NYMU P9010281.jpg

Descr Win length Fail length
1: GFP+terminator(19f) 854bp -
2: GFP(E0040) 720bp -
3: GFP(E0040) 720bp -
4: marker 100bp - -
5: Positive Control 1100 bp
6: Negative Control

2010.09.01

  • GFP*3 Plasmid Extraction 10:30
  • Digestionγ€€12:30 (13:20)
  • Run gel 15:30
NYMU P9070330.jpg

Descr Win length Fail length
1: - - -
2: - - -
3: marker 100bp - -
4: - - -
5: (GFP)E0040-2 745bp
6: (GFP)E0040-1 745bp
7: (GFP)J04630 856bp
Total:(*2) 50μl
template 2μl
VF+VR 2μl
dNTP 2μl
Buffer 5μl
ddH2O 39.25μl
Enzyme tag 0.25μl

  • RG1,RG2,RG3,PR1,PR2,PR3 3-in-1 10:00
  • Colony PCR 11:00~13:00
  • Run Gel


NYMU P9010283.jpg

"
Descr Win length Fail length
1: PFRV(1-a) 1158bp 294bp
2: PFRV(1-b) 1158bp 294bp
3: PFRV(2-a) 1158bp 294bp
4: PFRV(2-b) 1158bp 294bp
5: PFRV(3-a) 1158bp 294bp
6: PFRV(3-b) 1158bp 294bp
7: - - -
8: marker 100bp - -
9: PFRV 1366bp -
10: PFRV 1366bp -
11: PFRV 1366bp -
12: PFRV 1366bp
f 13: PFRV 1366bp
f 14: PFRV 1366bp
f 15: positive control 1100bp
w 16: negative control -
w
Total:(*2) 50μl temptime
template 2μl 9460s
VF+VR 2μl9415s
dNTP 2μl5520s
Buffer 5μl7290s
ddH2O 39.75μl72300s
Enzyme tag 0.25μl

2010.09.02

  • Liquid culture plasmid extraction 10:00
  • Nanodrop
  • Digestion 12:12
  • Purification
  • Nanodrop
  • Ligation
  • Transform 17:00

2010.09.03

  • 3-in-1 chose 4 colony
  • PCR 11.20
  • run gel
NYMU P9070329.jpg

Descr Win length Fail length
1: FRV1 1158bp 294bp
2: FRV2 1158bp 294bp
3: marker 1kb - -
4: FRV3 1158bp -
5: FRV4 1158bp 294bp
6: positive control 1100bp
7: negative control -
Total:(*2) 50μl
template 2μl
VF+VR 2μl
dNTP 2μl
Buffer 5μl
ddH2O 39.75μl
Enzyme tag 0.25μl

(the marker takes the wrong one,1kb marker is correct)

    • ligation failed
  • ligate again(insert+vector=8.5)
  • transform 16:40

2010.09.04

  • PCR 20:35pm (3 colony from 20100903 plate)
  • run gel(FRV1&2&3&+&-) 100V 35min
NYMU P9070327.jpg

Descr Win length Fail length
1: FRV1 1158bp 294bp
2: FRV2 1158bp 294bp
3: marker 100bp - -
4: FRV3 1158bp 294bp
5: positive control 1000bp -
6: negative control - -
Total:(*2) 50μl
template 2μl
VF+VR 2μl
dNTP 2μl
Buffer 5μl
ddH2O 39.75μl
Enzyme tag 0.25μl
  • discuss

2010.09.05

  • digest again(because we used XP's protocol for SP)
  • purification at 14:25
  • nanodrop at 15:00
    • we digest another tube (ribo2-2 SP) 15:50
  • ligation
  • transform 17:00

2010.09.06

  • Colony PCR 11:30
  • Run gel 14:30(2% argarose 100v 25 mins)
NYMU DSC05719.JPG

Descr Win length Fail length
1: positive control 1100bp -
2: negative control - -
3: 100bp marker - -
4: FRV 1158bp 1700bp
5: FRV 1158bp 1700bp
Total:(*2) 50μl
template 1μl
VF+VR 2μl
dNTP 2μl
Buffer 5μl
ddH2O 39.75μl
Enzyme tag 0.25μl

  • Take out the digest(sp)yesterday 10:30
  • digest purify
  • nanodrop(13.2ng/nl)
  • ligate 11:40~14:30
  • transform 17:00

2010.09.07

  • 3-in-1 10:30am
  • Run PCR 10:45am
NYMU DSC05720.JPG

Descr Win length Fail length
1: FRV 1158bp -
2: FRV 1158bp -
3: FRV 1158bp -
4: FRV 1158bp -
5: 100bp marker - -
6: FRV 1158bp -
7: FRV 1158bp 294bp
8: positive control 1100bp -
9: negative control - -
Total:(*2) 50μl
template 2μl
VF+VR
dNTP 2μl
Buffer 5μl
ddH2O 39.75μl
Enzyme tag 0.25μl

2010.09.08

  • plasmid extraction
  • digestion(FRV1(XP)) 11:10
    • nanodrop 14μl
  • ligation 14:00
  • Transformation 17:00

2010.09.09

  • Real PCR
NYMU R9.JPG


Total:(*2) 50μl
template 2μl
FP+RP 2μl
dNTP 2μl
Buffer 5μl
ddH2O 39.75μl
pfu 0.25μl
Temp Time
94 60s
94 15s
50.8 20s
74 45s
74 300s

2010.09.10

  • real PCR (temperature test)
  • run gel
NYMU DSC05722.JPG


Total:(*2) 50μl
template 2μl
FP+RP 2μl
dNTP 2μl
Buffer 5μl
ddH2O 39.75μl
pfu 0.25μl
Temp Time
94 60s
94 15s
50.8 20s
74 45s
74 300s
  • cut gel
  • gel extraction
  • digest(xp)
  • nanodrop
  • ligate
  • transform

  • real pcr for 2 tubes(in suitable temperature)
NYMU DSC05723.JPG


Total:(*2) 50μl
template 2μl
FP+RP 2μl
dNTP 2μl
Buffer 5μl
ddH2O 39.75μl
pfu 0.25μl
Temp Time
94 60s
94 15s
50.8 20s
74 45s
74 300s

2010.09.11

  • 3in1

  • two real tubes run gel and cut gel and gel extraction

NYMU DSC05725.JPG

2010.09.12

  • run gel(length isn't correct)

NYMU DSC05726.JPG

  • (real PCR) digestion 12:00
  • ligation 15:00
  • transform 19:40

2010.09.13

  • colony PCR 13:25 (3 in 1)
NYMU DSC05728.JPG


Total:(*2) 50μl
template 2μl
VR+VF 2μl
dNTP 2μl
Buffer 5μl
ddH2O 39.75μl
tag 0.25μl
Temp Time
94 60s
94 15s
55 20s
72 30s 30 cycle
72 300s

  • nanodrop
  • ligation 14:00
  • Colony PCR 15:00

2010.09.22

  • Real PCR
DSC05680.JPG
Total: 49μl X1.7
FP 1μl 1.7μl Theophylline Riboswitch Forward Primer
RP 1μl 1.7μl Theophylline Riboswitch Reverse Primer
dNTP 2μl 3.4μl
10XBuff. 5μl 8.5μl
pfu 0.25μl 0.425μl
ddH2O 39.75μl 67.575μl
PCR Protocol
94 60s
94 15s
52 20s
68 30s 35 cycles
68 300s


  • Digest ribo(xp) overnight
  • Ligate ribo with vector(xp)
  • Transform:23:30

2010.09.23

  • Ligate ribo with vector again
  • Transform

2010.09.24

  • 3-in-1 (ribo+vector)
  • colony PCR
DSC05682.JPG
Total: 49μl X1.9
VR+VF 2μl 3.8μl
dNTP 2μl 3.8μl
10XBuff. 5μl 9.5μl
tag 0.25μl 0.475μl
ddH2O 39.75μl 75.525μl
PCR Protocol
94 60s
94 15s
55 20s
72 30s 30 cycles
72 300s



  • Cut gel(ribo&pSB1A2(xp) digested)

DSC05683.JPG DSC05684.JPG

2010.09.26

  • 3-in-1(0925 plate)
  • colony PCR
DSC05682.JPG
Total: 49μl X2.5
VR+VF 2μl 5μl
dNTP 2μl 5μl
10XBuff. 5μl 12.5μl
tag 0.25μl 1.25μl
ddH2O 39.75μl 99.375μl
PCR Protocol
94 60s
94 15s
55 20s
72 30s 30 cycles
72 300s

2010.09.27

  • plasmid extraction(RV)
  • Digest RV 13:30
  • run gel

IMG 3189.JPG

There is GFP on vector, so it's wrong.

  • Digest ribo again overnight

2010.09.28

  • cut gel(ribo& vector)

DSC05689.JPG

  • Ligate ribo with vector
  • Transform

According to 0927 gel pic, 30th colony may be correct RV, so we take out second plate and incubate in LB liguid.

  • plasmid extraction 21:30
  • Digest RV(SP) overnight

2010.09.29

  • Ligate RV(sp) with GFP(BBa_J04630 already have terminator)
  • Transform

2010.09.30

  • Transform BBa_J04630

2010.10.01

  • 3-in-1
    • colony PCR
DSC05690.JPG
Total: 49μl X1.6
VR+VF 2μl 3.2μl
dNTP 2μl 3.2μl
10XBuff. 5μl 8μl
tag 0.25μl 0.4μl
ddH2O 39.75μl 63.6μl
PCR Protocol
94 60s
94 15s
55 20s
72 70s 30 cycles
72 300s

2010.10.02

  • BBa_J04630 plasmid extraction
  • Digest J04630(XP)
  • cut gel(GFP)

DSC05691.JPG

  • Ligate RV(SP) with GFP(XP)
  • Transform

2010.10.03

  • FRV(RV+GFP) 3-in-1
DSC05692.JPG
Total: 49μl X1.9
VR+VF 2μl 3.8μl
dNTP 2μl 3.8μl
10XBuff. 5μl 9.5μl
tag 0.25μl 0.475μl
ddH2O 39.75μl 75.525μl
PCR Protocol
94 60s
94 15s
55 20s
72 100s 30 cycles
72 300s

2010.10.04

  • FRV plasmid extraction
  • Digest FRV (XP)
  • cut gel & purify(FRV)

DSC05693.JPG

  • Ligate FRV with pLac(from ssrA group)
  • Transform

  • pLac plasmid extraction
  • Digest pLac(SP) overnight

2010.10.05

  • pLac digest purify(run gel with 3-in-1)
  • PFRV, GFP, RV 3-in-1
DSC05695.JPG
Total: 49μl X2.5
VR+VF 2μl 5μl
dNTP 2μl 5μl
10XBuff. 5μl 12.5μl
tag 0.25μl 0.625μl
ddH2O 39.75μl 99.375μl
PCR Protocol
94 60s
94 15s
55 20s
72 90s 30 cycles
72 300s



  • Digest pLac(SP) again overnight

2010.10.06

  • pLac cut gel

DSC05696.JPG

  • Ligate pLac(SP) with FRV(XP)
  • Transform 22:40

  • Digest FRV(XP) overnight

2010.10.07

  • FRV(XP) cut gel

DSC05697.JPG

  • Ligate FRV with pLac
  • Transform

2010.10.08

  • PFRV 3-in-1
DSC05698.JPG
Total: 49μl X3.8
VR+VF 2μl μl
dNTP 2μl 3.8μl
10XBuff. 5μl 9.5μl
tag 0.25μl 0.475mu;l
ddH2O 39.75μl 75.525μl
PCR Protocol
94 60s
94 15s
55 20s
72 100s 30 cycles
72 300s



  • Ligate FRV with pLac
  • Transform 22:15

2010.10.09

  • PFRV 3-in-1
DSC05699.JPG
Total: 49μl X3.8
VR+VF 2μl 7.6μl
dNTP 2μl 7.6μl
10XBuff. 5μl 17μl
tag 0.25μl 0.95μl
ddH2O 39.75μl 151.06μl
PCR Protocol
94 60s
94 15s
55 20s
72 90s 30 cycles
72 300s


  • colony PCR again
DSC05700.JPG
Total: 49μl X3.8
VR+VF 2μl 7.6μl
dNTP 2μl 7.6μl
10XBuff. 5μl 17μl
tag 0.25μl 0.95μl
ddH2O 39.75μl 151.06μl
PCR Protocol
94 60s
94 15s
55 20s
72 180s 30 cycles
72 300s


  • Ligate FRV with pLac again
  • Transform

2010.10.10

  • PFRV 3-in-1
DSC05702.JPG
Total: 49μl X3.2
VR+VF 2μl 6.4μl
dNTP 2μl 6.4μl
10XBuff. 5μl 15μl
tag 0.25μl 0.8μl
ddH2O 39.75μl 127.2μl
PCR Protocol
94 60s
94 15s
55 20s
72 180s 30 cycles
72 300s

length is correct!!!

  • F colony liquid culture
  • FRV liquid culture

  • Digest FRV(XP) again

2010.10.11

  • FRV&PFRV plasmid extraction
  • FRV cut gel

DSC05703.JPG

  • Digest PFRV(XP)&FRV&pSB1C3
  • Ligate PFRV with C3/ FRV with C3/ ribo with C3
  • Transform

2010.10.12

  • Ligate PFRV with C3/FRV with C3/ribo with C3 again
  • Transform 17:25

2010.10.13

  • PFRC&FRC&RC 3-in-1
DSC05704.JPG
Total: 49μl X1.7
VR+VF 2μl 3.4μl
dNTP 2μl 3.4μl
10XBuff. 5μl 8μl
tag 0.25μl 0.425μl
ddH2O 39.75μl 67.575μl
PCR Protocol
94 60s
94 15s
55 20s
72 90s 30 cycles
72 300s



  • Ligate PFRV,FRV,ribo with C3
  • Transform 00:50

  • Digest PFRV(XP)&RV(XP) again overnight

2010.10.14

  • PFRV&RV run gel

DSC05705.JPG


  • Ligate RV,FRV,PFRV with C3 again
  • Transform

2010.10.15

  • FRC,RC 3-in-1
DSC05706.JPG
Total: 49μl X2.5
VR+VF 2μl 5μl
dNTP 2μl 5μl
10XBuff. 5μl 12.5μl
tag 0.25μl 0.625μl
ddH2O 39.75μl 99.375μl
PCR Protocol
94 60s
94 15s
55 20s
72 90s 30 cycles
72 300s


  • 3-in-1 again
DSC05707.JPG
Total: 49μl X2.5
VR+VF 2μl 5μl
dNTP 2μl 5μl
10XBuff. 5μl 12.5μl
tag 0.25μl 0.625μl
ddH2O 39.75μl 99.375μl
PCR Protocol
94 60s
94 15s
55 20s
72 90s 30 cycles
72 300s

FRC and PFRC are correct, but RC is still incorrect

  • PFRC&FRC liquid culture

  • Ligate ribo with C3 again
  • Transform

2010.10.16

  • Theophylline solution
    • Take 1.8012g Theophylline into 15ml DMSO;concentration is 0.67M
  • put 15 μl into LB liquid, concentration is 2.5mM;

put 30 μl into LB liquid, concentration is 5mM


  • Ligate ribo(xp) with C3 again
  • Transform

2010.10.17

  • transform second plate of PFRC into agar plate

positive:C3 30μl+theophylline 0.0027g

negative:C3

  • PFRC&FRC plasmid extraction

2010.10.18

  • PFRC PCR test
DSC05708.JPG
Total: 49μl X1.3
VR+VF 2μl 2.6μl
dNTP 2μl 2.6μl
10XBuff. 5μl 6.5μl
tag 0.25μl 0.325μl
ddH2O 39.75μl 51.675μl
PCR Protocol
94 60s
94 15s
55 20s
72 90s 30 cycles
72 300s


  • Theophylline in DMSO
    • add 1 ml DMSO + Theophylline into 4 ml LB

  • Ligate ribo with C3 again
  • Transform

  • Digest ribo(XP)& C3(XP)
  • ribo & C3 cut gel

DSC05710.JPG

2010.10.19

  • Assay Test
O.D/2 O.D add to liquid
PFRC 2.503 5.006 166.5
LB 0 0
    • Because Theophylline entry into E. coli spend 1 hours at least, O.D= 0.0325
  • Assay:add 80 μl PFRC liquid into LB
Theophylline concentration Theophylline(0.67M) PFRC liquid(μl) Cm50(μl)
A 0 mM 0 80.4 4
B 0.5 mM 2.985 μl 80.4 4
C 1 mM 5.97 μl 80.4 4
D 2 mM 11.94 μl 80.4 4
E 4 mM 23.88 μl 80.4 4
F 5 mM 29.85 μpl 80,4 4

  • 10/18 RC 3-in-1
DSC05711.JPG
Total: 49μl X2.1
VR+VF 2μl 4.2μl
dNTP 2μl 4.2μl
10XBuff. 5μl 10.5μl
tag 0.25μl 0.525μl
ddH2O 39.75μl 83.475μl
PCR Protocol
94 60s
94 15s
55 20s
72 30s 30 cycles
72 300s


  • PFRC & C liquid culture

  • Ligate ribo with C3 again
  • Transform

2010.10.20

  • Assay
Theophylline concentration Theophylline(0.67M) PFRC liquid(μl) Cm50(μl)
O 0 mM 0 130 4
A 0.1 mM 0.597 μl 130 4
B 0.05 mM 1.4925 μl 130 4
C 0.5 mM 2.985 μl 130 4
D 1 mM 5.97 μl 130 4
E 2 mM 11.94 μl 130 4
F 4 mM 23.88 μpl 130 4
G 8 mM 47.76 μl 130 4

culture for 2 hours

  • Transform

2010.10.21

  • Assay
Theophylline concentration Theophylline(0.67M) PFRC liquid(μl) Cm50(μl)
O 0 μM 0 85.7 4
A 50 μM 0.2985 μl 85.7 4
B 100 μM 0.597 μl 85.7 4
C 200 μM 1.194 μl 85.7 4
D 500 μM 2.985 μl 85.7 4
E 1000 μM 5.97 μl 85.7 4
F 2000 μM 11.94 μl 85.7 4
G 4000 μM 23.88 μl 85.7 4
H 8000 μM 47.76 μl 85.7 4
I 10000 μM 59.7 μl 85.7 4
J 20000 μM 119.4 μl 85.7 4
K 40000 μM 238.8 μl 85.7 4
  • 10/20 RC 3-in-1
DSC05712.JPG
Total: 49μl X2.5
VR+VF 2μl 5μl
dNTP 2μl 5μl
10XBuff. 5μl 12.5μl
tag 0.25μl 1.25μl
ddH2O 39.75μl 99.375μl
PCR Protocol
94 60s
94 15s
55 20s
72 30s 30 cycles
72 300s


  • Digest ribo again

2010.10.22

  • Digest C3 from PFRC plasmid
  • Ligate ribo with C3
  • Transform

  • Preparing 0.1 M Theophylline solution in DMSO.
  • Assay
Theophylline concentration Theophylline(0.1M) PFRC liquid(μl) Cm50(μl)
O 0 μM 0 85.7 4
A 10 μM 0.4 μl 117.7 4
B 50 μM 2 μl 117.7 4
C 100 μM 4 μl 117.7 4
D 200 μM 8 μl 117.7 4
E 500 μM 20 μl 117.7 4
F 1000 μM 40 μl 117.7 4
G 2000 μM 80 μl 117.7 4
H 4000 μM 160 μl 117.7 4
I 8000 μM 320 μl 117.7 4
J 10000 μM 400 μl 117.7 4
K 20000 μM 800 μl 117.7 4
  • Modified experiment design
Theophylline concentration Theophylline(0.1M) PFRC liquid(μl)FRC liquid(μl) Cm50(μl)
O 0 μM 0 261 0 4
A 10 μM 0.4 μl 261 0 4
B 50 μM 2 μl 261 0 4
C 100 μM 4 μl 261 0 4
D 200 μM 8 μl 261 04
E 500 μM 20 μl 261 04
F 1000 μM 40 μpl 261 04
G 2000 μM 80 μl 261 04
H 4000 μM 160 μl 261 04
I 8000 μM 320 μl 261 04
J 10000 μM 400 μl 261 04
K 20000 μM 800 μl 261 04
NT 100 μM 4 μl 0 261 4
N 0 μM 0 μl 0 261 4

2010.10.24

  • ribo 3-in-1
DSC05713.JPG
Total: 49μl X0.9
VR+VF 2μl 1.8μl
dNTP 2μl 1.8μl
10XBuff. 5μl 4.5μl
tag 0.25μl 0.225μl
ddH2O 39.75μl 35.775μl
PCR Protocol
94 60s
94 15s
55 20s
72 30s 30 cycles
72 300s


  • ribo liquid culture
  • colony PCR
DSC05715.JPG
Total: 49μl X1.6
VR+VF 2μl 3.2μl
dNTP 2μl 3.2μl
10XBuff. 5μl 8μl
tag 0.25μl 0.8μl
ddH2O 39.75μl 63μl
PCR Protocol
94 60s
94 15s
55 20s
72 30s 30 cycles
72 300s


  • Assay
Theophylline concentration Theophylline(0.1M) Remove LB(μl) PFRC liquid(μl)FRC liquid(μl) Cm50(μl)
O 0 μM 0 325 325 0 4
A 10 μM 0.4 μl 325 325 0 4
B 50 μM 2 μl 327325 0 4
C 100 μM 4 μl 329 325 0 4
D 200 μM 8 μl 333325 04
E 500 μM 20 μl 345325 04
F 1000 μM 40 μpl 365325 04
G 2000 μM 80 μl 405325 04
H 4000 μM 160 μl 485325 04
I 8000 μM 320 μl 645325 04
J 10000 μM 400 μl 725 325 04
K 20000 μM 800 μl 1125 325 04
NT 100 μM 4 μl 504 0 500 4
N 0 μM 0 μl 500 0 500 4

  • Digest RV(XP)& FRC(XP)
  • Ligate ribo with C3
  • transform

  • PFRC&FRC liquid culture

2010.10.25

  • RC 3-in-1
DSC05716.JPG
Total: 49μl X2.4
VR+VF 2μl 4.8μl
dNTP 2μl 4.8μl
10XBuff. 5μl 12μl
tag 0.25μl 0.6μl
ddH2O 39.75μl 95.4μl
PCR Protocol
94 60s
94 15s
55 20s
72 30s 30 cycles
72 300s


  • FRC&PFRC&RC liquid culture


  • Assay
Theophylline concentration Theophylline(0.1M) Remove LB(μl) PFRC liquid(μl) FRC liquid(μl) Cm50(μl)
O 0 μM 0 151 147 0 4
A 50 μM 2 μl 153 147 0 4
B 500 μM 20 μl 171147 0 4
C 1000 μM 40 μl 191 147 0 4
D 4000 μM 160 μl 311147 0 4
E 10000 μM 400 μl 551147 0 4
NT 100 μM 4 μl 158 0150 4
N 0 μM 0 μl 154 0150 4

2010.10.26

  • Assay
Theophylline concentration Theophylline(0.1M) Remove LB(μl) PFRC liquid(μl) FRC liquid(μl) Cm50(μl)
O 0 μM 0 151 147 0 4
A 50 μM 2 μl 153 147 0 4
B 500 μM 20 μl 171147 0 4
C 1000 μM 40 μl 191 147 0 4
D 4000 μM 160 μl 311147 0 4
E 10000 μM 400 μl 551147 0 4
NT 100 μM 4 μl 158 0150 4
N 0 μM 0 μl 154 0150 4