MRNA Binding Experiments

From 2010.igem.org

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[[Image:NYMU_CIMG5632.JPG|left|400px|thumb|bend1~6 → apt.+ter.+RBS(XP)  //  maker  //  eIF4A on pSB1A2(XP) //  result of check is that (apt.+ter.)+(RBS) did not ligase, so we decided to ligase them again.]]
+
[[Image:NYMU_CIMG5632.JPG|left|400px|thumb|band1~6 → apt.+ter.+RBS(XP)  //  maker  //  eIF4A on pSB1A2(XP) //  result of check is that (apt.+ter.)+(RBS) did not ligase, so we decided to ligase them again.]]
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*run gel and gel extraction
*run gel and gel extraction
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[[Image:NYMU_CIMG5633.JPG|left|400px|thumb|bend1~10 → apt.+ter.(XP)  //  maker  //  eIF4A ]]
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[[Image:NYMU_CIMG5633.JPG|left|400px|thumb|band1~10 → apt.+ter.(XP)  //  maker  //  eIF4A ]]
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*ligation of apt.+ter(XP)+(RBS(SP)  
*ligation of apt.+ter(XP)+(RBS(SP)  
*transformation
*transformation
 +
=2010.08.31=
=2010.08.31=
*3 in 1 of '''apt+term+RBS'''
*3 in 1 of '''apt+term+RBS'''
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[[Image:NYMU_CIMG5635.JPG|left|400px|thumb|mut.2 and3 didn't have bends, so we PCR test again. ]]
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[[Image:NYMU_CIMG5635.JPG|left|400px|thumb|mut.2 and3 didn't have bands, so we PCR test again. ]]
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[[image:NYMU_2010_10_28.png|left|400px|thumb| ]]
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=2010.09.10=
=2010.09.10=
*3 in 1 of pSB1C3+pLac+RBS+apt+term and pSB1C3+RBS+apt+term(because LB tube did not be put for cultivating><)
*3 in 1 of pSB1C3+pLac+RBS+apt+term and pSB1C3+RBS+apt+term(because LB tube did not be put for cultivating><)
Line 1,380: Line 1,385:
=2010.09.11=
=2010.09.11=
*plasmid extraction of pSB1C3+pLac+RBS+aptamer+terminator and pSB1C3+RBS+aptamer+terminator succeed! Have two new 2010 biobricks!
*plasmid extraction of pSB1C3+pLac+RBS+aptamer+terminator and pSB1C3+RBS+aptamer+terminator succeed! Have two new 2010 biobricks!
-
*The PCR of yesterday failed. So, do it again. And the result of the PCR today sill failed.(no bends><), we still figure what the problem is.
+
*The PCR of yesterday failed. So, do it again. And the result of the PCR today sill failed.(no bands><), we still figure what the problem is.
*The plate of pSB1C3+aptamer+terminator have no colonies, so ligate again.
*The plate of pSB1C3+aptamer+terminator have no colonies, so ligate again.
*digestion of pSB1C3(XP)  
*digestion of pSB1C3(XP)  
Line 1,416: Line 1,421:
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[[image:NYMU_2010_9_11.png|left|400px|thumb| ]]
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=2010.09.12=
=2010.09.12=
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[[image:NYMU_2010_9_14.png|left|400px|thumb| ]]
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=2010.9.15=
=2010.9.15=
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[[image:NYMU_2010_9_16.png|left|400px|thumb| ]]
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=2010.9.17=
=2010.9.17=
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[[image:NYMU_2010_9_21.png|left|400px|thumb| ]]
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[[image:NYMU_2010_9_21_1.png|left|400px|thumb| ]]
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=2010.9.22=
=2010.9.22=
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[[image:NYMU_2010_9_22.png|left|400px|thumb| ]]
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[[image:NYMU_2010_9_22_1.png|left|400px|thumb| ]]
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=2010.9.23=
=2010.9.23=
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[[image:NYMU_2010_9_24.png|left|400px|thumb| ]]
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[[image:NYMU_2010_9_24_1.png|left|400px|thumb| ]]
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=2010.09.25=
=2010.09.25=
*plasmid extration of  
*plasmid extration of  
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[[image:NYMU_2010_9_25.png|left|400px|thumb| ]]
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[[image:NYMU_2010_9_25_1.png|left|400px|thumb| ]]
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=2010.09.26=
=2010.09.26=
*colony PCR of GA(X2),GB(X2),RA(X2),RB(X2),on pSB1A2
*colony PCR of GA(X2),GB(X2),RA(X2),RB(X2),on pSB1A2
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*digestion and gel extration of pSB1C3(XP), pSB1A2(XP)
*digestion and gel extration of pSB1C3(XP), pSB1A2(XP)
 +
[[image:NYMU_2010_9_26.png|left|400px|thumb| ]]
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=2010.09.27=
=2010.09.27=
*plasmid extraction of GFP-e-A-2,GFP-e-B-2,RFP-e-A-2,RFP-e-B-2
*plasmid extraction of GFP-e-A-2,GFP-e-B-2,RFP-e-A-2,RFP-e-B-2
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*transformation.
*transformation.
 +
[[image:NYMU_2010_9_27.png|left|400px|thumb| ]]
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[[image:NYMU_2010_9_27_1.png|left|400px|thumb| ]]
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[[image:NYMU_2010_9_27_2.png|left|400px|thumb| ]]
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=2010.09.28=
=2010.09.28=
*3 in 1 of colony PCR of GA,GB,RA,RB
*3 in 1 of colony PCR of GA,GB,RA,RB
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[[image:NYMU_2010_9_28.png|left|400pixel|thumb| ]]
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=2010.09.29=
=2010.09.29=
*plasmid extractio of GA(1),GB(2),RA(1),RB(2)
*plasmid extractio of GA(1),GB(2),RA(1),RB(2)
Line 2,506: Line 2,579:
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[[image:NYMU_2010_10_01.png|left|400px|thumb| ]]
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=2010.10.02=
=2010.10.02=
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| 72
| 72
| 300s
| 300s
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 +
*ligation of
 +
(1)JGeA+terminator
 +
 +
(2)JReA
 +
 +
(3)JReB
 +
 +
(4)C3+GeA
 +
 +
(5)C3+GeB
 +
 +
(6)C3+ReA
 +
 +
(7)C3+ReB
 +
 +
(8)C3+eIF4A
 +
**ligation protocol of GA,GB
 +
 +
{| border=1
 +
|-
 +
! Total !! 10&mu;l
 +
|-
 +
| buffer || 1&mu;l
 +
|-
 +
| ligase || 0.5&mu;l
 +
|-
 +
| DNA1(vector) || 1&mu;l
 +
|-
 +
| DNA2(~) || 2&mu;l
 +
|-
 +
| ddH2O || 5.5&mu;l
|-
|-
|}
|}
=2010.10.21=
=2010.10.21=
 +
*ligation of J13002 ReB,pSB1C3+ReA/ReB/GeA/GeB/eIF4A
 +
**ligation protocol
 +
 +
{| border=1
 +
|-
 +
! Total !! 10&mu;l
 +
|-
 +
| buffer || 1&mu;l
 +
|-
 +
| ligase || 0.5&mu;l
 +
|-
 +
| DNA1(vector) || 1&mu;l
 +
|-
 +
| DNA2(~) || 2&mu;l
 +
|-
 +
| ddH2O || 5.5&mu;l
 +
|-
 +
|}
 +
=2010.10.22=
=2010.10.22=
 +
*ligation of J13002+ReA/ReB,pSB1C3+eIF4A/GeA/GeB/ReA/ReB
 +
**ligation protocol
 +
 +
{| border=1
 +
|-
 +
! Total !! 10&mu;l
 +
|-
 +
| buffer || 1&mu;l
 +
|-
 +
| ligase || 0.5&mu;l
 +
|-
 +
| DNA1(vector) || 1&mu;l
 +
|-
 +
| DNA2(~) || 2&mu;l
 +
|-
 +
| ddH2O || 5.5&mu;l
 +
|-
 +
|}
 +
=2010.10.23=
=2010.10.23=
 +
*3in1 of JGeA, JReA, JReB
 +
**colony PCR protocol
 +
 +
{| border=1
 +
|-
 +
! Total: !! 50&mu;l || X5&mu;l
 +
|-
 +
| Template || 1&mu;l
 +
|-
 +
| FP || 1&mu;l || 
 +
|-
 +
| RP || 1&mu;l || 
 +
|-
 +
| dNTP || 2&mu;l || 10&mu;l 
 +
|-
 +
| 10XBuff. || 5&mu;l || 25&mu;l
 +
|-
 +
| tag || 0.25&mu;l || 1.25&mu;l 
 +
|-
 +
| ddH2O || 39.75&mu;l || 198.75&mu;l
 +
|}
 +
{|
 +
|-
 +
|
 +
{| border="1"
 +
|-
 +
! PCR Protocol !!
 +
|-
 +
| 94
 +
| 60s
 +
|-
 +
| 94
 +
| 15s
 +
|-
 +
| 55
 +
| 20s
 +
|-
 +
| 72
 +
| 100s
 +
| 30 cycles
 +
|-
 +
| 72
 +
| 300s
 +
|-
 +
|}
 +
=2010.10.24=
=2010.10.24=
 +
*3in1 of JReA, JReB, C3+ReB/eIF4A/GA/GB/RA
 +
**colony PCR protocol
 +
 +
{| border=1
 +
|-
 +
! Total: !! 50&mu;l || X6.6&mu;l
 +
|-
 +
| Template || 1&mu;l
 +
|-
 +
| FP || 1&mu;l || 
 +
|-
 +
| RP || 1&mu;l || 
 +
|-
 +
| dNTP || 2&mu;l || 13.2&mu;l 
 +
|-
 +
| 10XBuff. || 5&mu;l || 33&mu;l
 +
|-
 +
| tag || 0.25&mu;l || 1.65&mu;l 
 +
|-
 +
| ddH2O || 39.75&mu;l || 262.35&mu;l
 +
|}
 +
{|
 +
|-
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|
 +
{| border="1"
 +
|-
 +
! PCR Protocol !!
 +
|-
 +
| 94
 +
| 60s
 +
|-
 +
| 94
 +
| 15s
 +
|-
 +
| 55
 +
| 20s
 +
|-
 +
| 72
 +
| 100s
 +
| 30 cycles
 +
|-
 +
| 72
 +
| 300s
 +
|-
 +
|}
 +
[[image:DSC05773.JPG|left|400px|thumb| ]]
 +
*digestion of JGeAT,JGeT
 +
**digestion protocol
 +
{| border=1
 +
|-
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! Total !! 20&mu;l
 +
|-
 +
| DNA || 16&mu;l
 +
|-
 +
| Digestion buffer || 2&mu;l
 +
|-
 +
| Enzyme 1 (X) || 1&mu;l
 +
|-
 +
| Enzyme 2 (P) || 1&mu;l
 +
|-
 +
|}
 +
=2010.10.25=
=2010.10.25=
=2010.10.26=
=2010.10.26=
-
=2010.10.27=
+
*ligation of pSB1C3+eIF4A/GeA/ReA/ReB
-
=2010.10.28=
+
**ligation protocol
 +
 
 +
{| border=1
 +
|-
 +
! Total !! 10&mu;l
 +
|-
 +
| buffer || 1&mu;l
 +
|-
 +
| ligase || 0.5&mu;l
 +
|-
 +
| DNA1(vector) || 1&mu;l
 +
|-
 +
| DNA2(~) || 2&mu;l
 +
|-
 +
| ddH2O || 5.5&mu;l
 +
|-
 +
|}
 +
 
 +
*3in1 of pSB1C3+GeA/ReA/ReB
 +
 
{{:Team:NYMU-Taipei/Footer}}
{{:Team:NYMU-Taipei/Footer}}

Latest revision as of 03:53, 28 October 2010

Contents

2010.08.09

  • PCR of split GFP(A.B)
    • PCR of split GFP (test/+tag/5 tubes)
Total: 50μl X2.8μl FP RP Template
Template 1μl 5.6μl negative VF2 VR
FP 1μl positive VF2 VR complete GFP
RP 1μl GFP_A+B GFP_split_A_FP GFP_split_B_RP complete GFP
dNTP 2μl 5.6μl GFP_A GFP_split_A_FP GFP_split_A_RP complete GFP
10XBuff. 5μl 14μl GFP_B GFP_split_B_FP GFP_split_B_RP complete GFP
taq 0.25μl 0.7μl
ddH2O 39.75μl 111.3μl
PCR Protocol
94 60s
94 15s
55 20s
72 70s 30 cycles
72 300s


    • PCR of split GFP (use pfu/4 tubes)
Total: 50μl X2μl FP RP Template
Template 1μl 2μl negative VF2 VR
FP 1μl positive VF2 VR complete GFP
RP 1μl GFP_A GFP_split_A_FP GFP_split_A_RP complete GFP
dNTP 4μl 8μl GFP_B GFP_split_B_FP GFP_split_B_RP complete GFP
10XBuff. 5μl 10μl
pfu 1μl 2μl
ddH2O 37μl 74μl
PCR Protocol
94 120s
94 30s
55 30s
72 150s 35 cycles
72 300s

2010.08.10

  • PCR of GFP (A+B)
    • To make sure the original GFP+eIF4A can emit fluorescence.
    • Gel extration >> purification of split GFP(A.B)
    • PCR of GFP(A+B) (use pfu/3 tubes)
Total: 50μl X1.5μl FP RP
Template 1μl negative
FP 1μl positive VF2 VR
RP 1μl GFP(A+B) GFP_split_A_FP GFP_split_B_RP
dNTP 4μl 6μl
10XBuff. 5μl 7.5μl
pfu 1μl 1.5μl
ddH2O 37μl 56.25μl
PCR Protocol
94 120s
94 30s
55 30s
72 150s 35 cycles
72 300s

2010.08.12

  • PCR of aptamer
    • PCR >> Gel extraction >> purification
    • PCR (use pfu/3 tubes)
Total: 50μl X2μl FP RP length
FP 1μl negative
RP 1μl positive(pTet-1) VF2 VR 292
dNTP 4μl 8μl aptamer aptamer_FP aptamer_RP 96
10XBuff. 5μl 10μl
pfu 1μl 2μl
ddH2O 38μl 76μl
PCR Protocol
94 120s
94 30s
55 30s
72 40s 35 cycles
72 300s

2010.08.13

  • digestion,PCR purification,ligation and transformation(aptamer on pSB1A2)
    • digestion of aptamer (XP) >> PCR purification >> ligation(aptamer+pSB1A2 from YFP) >> transfomation
    • digestion protocol
Total 30μl
DNA 26μl
Digestion buffer 2μl
Enzyme 1 (X) 1μl
Enzyme 2 (P) 1μl
    • ligation protocol
Total 10μl length
ligase 0.5μl
ligation buffer 1μl
DNA1(aptamer(XP)) 8μl 88
DNA2(pSB1A2) 0.5μl 2100

2010.08.16

  • 3 in 1 of aptamer on pSB1A2
    • PCR protocol for test(5tubes)
Total: 50μl X3.6μl
Template 1μl
FP 1μl 3.6μl
RP 1μl 3.6μl
dNTP 2μl 7.2μl
10XBuff. 5μl 18μl
taq 0.25μl 0.9μl
ddH2O 39.75μl 143.1μl
PCR Protocol
94 120s
94 15s
55 20s
72 30s 30 cycles
72 300s

2010.08.17

  • plasmid extraction of aptamer on plasmid and digestion aptamer SP
    • digestion protocol
Total 20μl
DNA 16μl
Digestion buffer 2μl
Enzyme 1 (S) 1μl
Enzyme 2 (P) 1μl

2010.08.18

  • gel extraction >> purification >> ligation >> transformation of aptamer(SP)
    • Transformation failed, so we digest again on 8/20. (no colonies)
    • ligation protocol
Total 10μl length
ligase 0.5μl
ligation buffer 1μl
DNA1(aptamer(SP)) 3μl 332
DNA2(terminator) 5.5μl 129

2010.08.20

  • digestion of aptamer(SP) and terminator(XP)
    • Do gel extraction, and when running electrophoresis we found that digestion failed so we restart to digest again.

2010.08.21

  • gel extraction and purification of aptamer(SP)
    • We checked the result of digestion and aptamer succeed but terminator failed.
    • continued to purify aptamer(SP), and ligation aptamer(SP) and terminator(XP,from SSrA)
    • transformation

2010.08.22

  • 3 in 1 of aptamer+terminator
    • colony PCR and run electrophoresis
    • negative was contaminated

2010.08.23

  • colony PCR of aptamer+terminator and plasmid extraction
    • check result of 8/22 but contaminated
    • restart to digest terminator(XP) and aptamer+terminator(XP)

2010.08.24

  • aptamer purification and eIF4A PCR
    • We run electrophoresis to check and terminator(XP) succeed, aptamer+terminator(XP) failed.
    • eIF4A PCR(use tag)

2010.08.25

  • 3 in 1 of aptamer and eIF4A electrophoresis(and design primer)

2010.08.26

  • plasmid extraction,digestion and purification of apt.+ter.
    • result of extraction → 3.4.5 be poluted,so use 1.2. for digest
    • digestion protocol(apt.+ter.)(XP)


Total 20μl
DNA 16μl
Digestion buffer 2μl
Enzyme 1 (X) 1μl
Enzyme 2 (P) 1μl
  • electrophoresis of tag Gradient(eIF4A) and apt.+ter.
left → tube 1. right → tube 2.
the result of check → It is possible for VR.VF2 to be polluted.





























  • also purification of RBS
  • PCR of complete eIF4A → purification→ digestion(XP)→ ligation with pSB1A2(XP) → transformation
  • real PCR solution of complete eIF4A(X2)(take out at 18:50)
Total: 50μl X2μl FP RP Template
Template 1μl negative VF2 VR
FP 1μl 2μl positive VF2 VR complete GFP
RP 1μl 2μl
dNTP 4μl 8μl
10XBuff. 5μl 10μl
pfu 1μl 2μl
ddH2O 37μl 74μl
PCR Protocol
94 120s
94 15s
53.6 20s
72 90s 35 cycles
72 60s
  • ligation protocol(10mins)
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1(eIF4A[XP]) 3μl
DNA2(pSB1A2[XP]) 1μl
ddH2O 4.5μl
  • transform(competent cell 30ul)
  • ligation of [apt.+ter.]+RBS
    • ligation protocol
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1(apt.+ter[XP]) 7.7μl
DNA2(RBS[SP]) 0.8μl

2010.08.27

  • colony PCR of apt+ter+RBS
  • colony PCR of eIF4A+pSB1A2
result of both of the PCR failed, it is possible for the negative control to be contaminated. We change the system of colony PCR, and PCR the eIF4A+pSB1A2 again.

















2010.08.28

  • colony PCR check
NYMU CIMG5631.JPG
















2010.08.29

  • plasmid extraction of eIF4A on pSB1A2 and apt.+ter.+RBS
  • digestion of eIF4A on pSB1A2(XP)[check], apt.+ter.+RBS(XP), pLac(SP) and eIF4A(XP)
    • digestion protocol of eIF4A on pSB1A2(XP) and apt.+ter.+RBS(XP)
Total 20μl
DNA 16μl
Digestion buffer 2μl
Enzyme 1 (X) 1μl
Enzyme 2 (P) 1μl
band1~6 → apt.+ter.+RBS(XP) // maker // eIF4A on pSB1A2(XP) // result of check is that (apt.+ter.)+(RBS) did not ligase, so we decided to ligase them again.

















    • digestion protocol of pLac(SP) and eIF4A(XP)
Total 20μl
DNA 10μl
Digestion buffer 2μl
Enzyme 1 (S) 1μl
Enzyme 2 (P) 1μl
ddH2O 6μl
  • purification of eIF4A(XP) and pLac(SP)
  • ligation of (apt.+ter/XP)+(RBS/SP)
    • ligation protocol
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1(apt.+ter[XP]) 7.7μl
DNA2(RBS[SP]) 0.8μl
  • transformation of the above one

2010.08.30

  • We discover that eIF4A sequence has 2 PstI enzyme cutting sites, so we design primer and make a eIF4A sequence by ourselves.
  • There are no colonies on the transformation plate yesterday(apt.+ter(XP))+(RBS(SP)), so we digest the apt.+ter/XP and RBS/SP for the beginning.
    • digestion protocol of apt.+ter(XP)
Total 20μl
DNA 16μl
Digestion buffer 2μl
Enzyme 1 (X) 1μl
Enzyme 2 (P) 1μl
    • digestion protocol of RBS(SP)
Total 20μl
DNA 10μl
Digestion buffer 2μl
Enzyme 1 (X) 1μl
Enzyme 2 (P) 1μl
ddddH2O 6μl
  • run gel and gel extraction
band1~10 → apt.+ter.(XP) // maker // eIF4A
















  • ligation of apt.+ter(XP)+(RBS(SP)
  • transformation

2010.08.31

  • 3 in 1 of apt+term+RBS
  • colony PCR of apt+term+RBS
Total: 50μl X2μl FP RP Template
Template 1μl negative VF2 VR
FP 1μl 5μl positive VF2 VR complete GFP
RP 1μl 5μl
dNTP 2μl 10μl
10XBuff. 5μl 25μl
taq 0.25μl 1.25μl
ddH2O 39.75μl 198.75μl
PCR Protocol
94 60s
94 15s
55 20s
72 40s 30 cycles
72 300s
tube 4 ligase successfully.















  • colony PCR(for test) of eIF4A 1,2,3(mutant part 1.2.3) >>>> 55'C
Total: 50μl X2.5μl FP RP Template
Template 1μl 5.6μl negative
FP 1μl positive eIF4A-split-A-FP eIF4A-split-B-RP eIF4A/C212-2
RP 1μl mutant1 eIF4A-split-A-FP eIF4A-mutant-1-RP eIF4A/C212-2
dNTP 2μl 5μl mutant2 eIF4A-mutant-1-FP eIF4A-mutant-2-RP eIF4A/C212-2
10XBuff. 5μl 12.5μl mutant3 eIF4A-mutant-2-FP eIF4A-split-B-RP eIF4A/C212-2
taq 0.25μl 0.625μl
ddH2O 39.75μl 99.375μl
PCR Protocol
94 60s
94 15s
53.6 20s
72 80s 30 cycles
72 300s
mut.2 and3 didn't have bands, so we PCR test again.















  • colony PCR(for test) of eIF4A 1,2,3(mutant part 1.2.3)>>>>>>>> for the second time! >>>> 51'C

2010.09.01

  • plasimd extraction of apt.+term.+RBS(X2 tubes)
  • digestion of apt.+term.+RBS(XP) and use our own pLac(SP) for ligation
    • digestion protocol of apt.+term.+RBS(XP)
Total 20μl
DNA 16μl
Digestion buffer 2μl
Enzyme 1 (X) 1μl
Enzyme 2 (P) 1μl
  • run gel >> gel extraction of apt.+term.+RBS(XP)
  • purification of apt.+term.+RBS(XP)>> ligation >> transformation(9/1 18:00)
    • ligation protocol
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1(apt.+ter+RBS[XP]) 6.7μl length:235
DNA2(pLac[SP]) 1.8μl length:200
  • eIF4A PCR test result(yesterday)
all succeed.YA!















  • PCR of eIF4A(pfu)
Total: 50μl X2μl FP RP Template length
Template 1μl negative
FP 1μl positive eIF4A-split-A-FP eIF4A-split-B-RP eIF4A/C212-2 1216
RP 1μl mutant1 eIF4A-split-A-FP eIF4A-mutant-1-RP eIF4A/C212-2 254
dNTP 4μl 8μl mutant2 eIF4A-mutant-1-FP eIF4A-mutant-2-RP eIF4A/C212-2 191
10XBuff. 5μl 10μl mutant3 eIF4A-mutant-2-FP eIF4A-split-B-RP eIF4A/C212-2 811
pfu 1μl 2μl
ddH2O 37μl 74μl
PCR Protocol
94 120s
94 30s
51 30s
72 160s 35 cycles
72 60s

2010.09.02

  • 3 in 1 of (apt.+term.+RBS)+(pLac) >> PCR >> run gel for check
    • length calculate
Total 661
aptamer+scar 58+8
terminator+scar 129+8
RBS+scar 12+8
pLac 200
VR-VF2 238
    • PCR protocol for test(6tubes)
Total: 50μl X3μl FP RP Template
Template 1μl negative VF2 VR
FP 1μl 3μl positive VF2 VR complete GFP
RP 1μl 3μl
dNTP 2μl 6μl
10XBuff. 5μl 15μl
taq 0.25μl 0.75μl
ddH2O 39.75μl 119.25μl
PCR Protocol
94 60s
94 15s
55 20s
72 60s 30 cycles
72 300s
    • result
all succeed.(tube1 was taken by SsrA group as negative.= =















  • run gel of eIF4A mutant1.2.3 >> gel extraction >> purification >> PCR of mutant1+2 and mutant2+3
    • run gel result of mutant1.2.3
all succeed.















    • PCR protocol for test(4tubes)
Total: 50μl X2μl FP RP Template length
Template 1μl negative
FP 1μl positive eIF4A-split-A-FP eIF4A-split-B-RP eIF4A/C212-2 1216
RP 1μl mutant1+2 eIF4A-split-A-FP eIF4A-mutant-2-RP eIF4A/C212-2 445-8
dNTP 4μl 8μl mutant2+3 eIF4A-mutant-1-FP eIF4A-split-B-RP eIF4A/C212-2 1002-8
10XBuff. 5μl 10μl
pfu 1μl 2μl
ddH2O 37μl 74μl
PCR Protocol
94 120s
94 30s
51 30s
72 160s 35 cycles
72 600s
    • result
all failed.So we do again at 9/3.















2010.09.03

  • plasimd extraction of aptamer+terminator+RBS+pLac, and the aptamer part have been done!!! (can use any other promotor)
  • PCR of eIF4A mutant1+2, mutant2+3 again >> run gel >> gel extraction >> purification
    • protocol as yesterday.
    • result
all succeed.















  • PCR of eIF4A mutant(1+2)+3, mutant1+(2+3)
    • PCR protocol
Total: 50μl X2μl FP RP Template length
Template 1μl negative
FP 1μl positive eIF4A-split-A-FP eIF4A-split-B-RP eIF4A/C212-2 1216
RP 1μl mutant(1+2)+3 eIF4A-split-A-FP eIF4A-split-B-RP mutant(1+2) 1216
dNTP 4μl 8μl mutant1+(2+3) eIF4A-split-A-FP eIF4A-split-B-RP mutant(2+3) 1216
10XBuff. 5μl 10μl
pfu 1μl 2μl
ddH2O 37μl 74μl
PCR Protocol
94 120s
94 30s
51 30s
72 160s 35 cycles
72 600s

2010.09.04

2010.09.05

2010.09.06

2010.09.07

2010.09.08

  • 3 in 1 of pSB1C3+aptamer/+apt+term/+RBS+apt+term/+pLac+RBS+apt+term
  • colony PCR (12 tubes)
  • pSB1C3+RBS+apt+term and pSB1C3+pLac+RBS+apt+term need to be transformed again( Because there are no coloines on plate.)

2010.09.09

  • elute dried DNA(pGEX-eIF4A) from kind Pro.C.Proud(use 60ul TE buffer and put at 4'C overnight)
    • pGEX is anti ampicillin.
  • plasmid extraction of pSB1C3+aptamer and pSB1C3+pLac
  • run gel of (1) pSB1C3+aptamer+terminator (2) pSB1C3+pLac (3) posotive (4) negative
    • result of the above: all succeed!!So, pSB1C3+pLac can be put on our 2010 biobrick!(LB tube of pSB1C3+aptamer have nothing.)
  • ligation of (pLac+RBS+aptamer+terminator)+pSB1C3 >> transformation
    • ligaiton protocol
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1(pSB1C3) 2μl
DNA2(pLac+RBS+aptamer+terminator) 3μl
ddH2O 3.5μl
NYMU 2010 10 28.png

2010.09.10

  • 3 in 1 of pSB1C3+pLac+RBS+apt+term and pSB1C3+RBS+apt+term(because LB tube did not be put for cultivating><)
  • ligation of (pSB1C3)+(apt+term) >> transformation
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1(pSB1C3) 1μl
DNA2(pLac+RBS+aptamer+terminator) 1.5μl
ddH2O 6μl
  • PCR colony of (1.2) pSB1C3+pLac+RBS+apt+term (3) pSB1C3+aptamer (4) pSB1C3+RBS+apt+term
    • PCR protocol for test(6tubes)
Total: 50μl X3μl FP RP Template
Template 1μl negative VF2 VR
FP 1μl 3μl positive VF2 VR pLac+RBS+apt+term
RP 1μl 3μl
dNTP 2μl 6μl
10XBuff. 5μl 15μl
taq 0.25μl 0.75μl
ddH2O 39.75μl 119.25μl
PCR Protocol
94 60s
94 15s
55 20s
72 60s 30 cycles
72 300s

2010.09.11

  • plasmid extraction of pSB1C3+pLac+RBS+aptamer+terminator and pSB1C3+RBS+aptamer+terminator succeed! Have two new 2010 biobricks!
  • The PCR of yesterday failed. So, do it again. And the result of the PCR today sill failed.(no bands><), we still figure what the problem is.
  • The plate of pSB1C3+aptamer+terminator have no colonies, so ligate again.
  • digestion of pSB1C3(XP)
    • digestion protocol
Total 20μl
DNA 10μl
Digestion buffer 2μl
Enzyme 1 (X) 1μl
Enzyme 2 (P) 1μl
ddH2O 6μl
  • ligation of pSB1C3+aptamer+terminator >> transformation
    • ligaiton protocol
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1(pSB1C3) 3.5μl
DNA2(aptamer+terminator) 1.5μl
ddH2O 3.5μl
NYMU 2010 9 11.png

2010.09.12

  • colony PCR of complete eIF4A
    • real PCR protocol(taq)
Total: 50μl X1.5μl FP RP Template
Template 1μl negative VF2 VR
FP 1μl 1.5μl positive VF2 VR complete GFP
RP 1μl 1.5μl
dNTP 2μl 3μl
10XBuff. 5μl 7.5μl
taq 0.25μl 0.375μl
ddH2O 39.75μl 596.25μl


PCR Protocol
94 60s
94 15s
51 20s
72 80s 30 cycles
72 300s

2010.09.13

  • transform (pGEX-eIF4A)(because the concentration of template from C.Proud is too low)
  • PCR(3 tubes) Test


Total: 50μl X1.5μl FP RP Template length
Template 1μl negative
FP 1μl positive VF2 VR GFP
RP 1μl eIF4A(0.3+water0.2) eIF4A-split-A-FP eIF4A-split-B-RP eIF4A 1216
dNTP 4μl 3μl
10XBuff. 5μl 7.5μl
pfu 1μl 0.375μl
ddH2O 37μl 59.625μl
PCR Protocol
94 60s
94 15s
55 20s
72 80s 30 cycles
72 300s

2010.9.14

  • 3in1 of pGEX-eIF4A
    • colony PCR(6tubes)
Total: 50μl X3μl FP RP Template length
Template 1μl negative
FP 1μl 3μl positive(eIF4A) VF2 VR GFP
RP 1μl 3μl pGEX-eIF4A(1) eIF4A-split-A-FP eIF4A-split-B-RP eIF4A 1216
dNTP 2μl 6μl pGEX-eIF4A(2) eIF4A-split-A-FP eIF4A-split-B-RP eIF4A 1216
10XBuff. 5μl 15μl pGEX-eIF4A(3) eIF4A-split-A-FP eIF4A-split-B-RP eIF4A 1216
taq 0.25μl 0.75μl pGEX-eIF4A(4) eIF4A-split-A-FP eIF4A-split-B-RP eIF4A 1216
ddH2O 39.75μl 119.25μl
PCR Protocol
94 60s
94 15s
55 20s
72 80s 30 cycles
72 300s
NYMU 2010 9 14.png

2010.9.15

  • real PCR(5tubes) of split eIF4A mutI, mutII, mutIII
Total: 50μl X2.5μl FP RP Template length
Template 1μl negative
FP 1μl 2.5μl positive(eIF4A) eIF4A-split-A-FP eIF4A-split-B-RP eIF4A 1216
RP 1μl 2.5μl mutI(1) eIF4A-split-A-FP eIF4A-mutI-RP eIF4A 258
dNTP 2μl 10μl mutII(2) eIF4A-mutI-FP eIF4A-mutII-RP eIF4A 191
10XBuff. 5μl 12.5μl mutIII(3) eIF4A-mutII-FP eIF4A-split-B-RP eIF4A 811
MgSO4 4μl 10μl
KOD 1μl 2.5μl
ddH2O 39.75μl 82.5μl
PCR Protocol
94 120s
94 30s
55 30s
72 160s 35 cycles
72 600s
Total: 50μl X2μl FP RP Template length
Template 1μl negative
FP 1μl 2μl positive(eIF4A) eIF4A-split-A-FP eIF4A-split-B-RP eIF4A 1216
RP 1μl 2μl mutI+mutII eIF4A-split-A-FP eIF4A-mutII-RP mutI+mutII 424
dNTP 2μl 4μl mutII+mutIII eIF4A-mutI-FP eIF4A-split-B-RP mutII+mutIII 983
10XBuff. 5μl 10μl mutI+mutII+mutIII eIF4A-split-A-FP eIF4A-split-B-RP mutI+mutII+mutIII 1216
pfu 0.25μl 0.5μl
ddH2O 39.75μl 79.5μl
PCR Protocol
94 120s
94 30s
55 30s
72 160s 35 cycles
72 600s

2010.9.16

  • run gel→gel extraction
  • real PCR
  • real PCR protocol
Total: 50μl X2μl FP RP Template length
Template 1μl negative
FP 1μl 2μl positive(eIF4A) eIF4A-split-A-FP eIF4A-split-B-RP eIF4A 1216
RP 1μl 2μl mut(I+II)+III eIF4A-split-A-FP eIF4A-split-B-RP mutI+mut(II+III) 1216
dNTP 2μl 4μl mutI+(II+III) eIF4A-split-A-FP eIF4A-split-B-RP mutI+mutII+mutIII 1216
10XBuff. 5μl 10μl mut(I+II+III) eIF4A-split-A-FP eIF4A-split-B-RP mutI+mutII+mutIII 1216
pfu 0.25μl 0.5μl
ddH2O 39.75μl 79.5μl
PCR Protocol
94 120s
94 30s
55 30s
72 160s 35 cycles
72 600s
  • digestion of eIF4A on pGEX
    • digestion protocol
Total 20μl
DNA(pGEX-eIF4A1) 2μl
Digestion buffer 2μl
Enzyme 1 (X) 1μl
Enzyme 2 (P) 1μl
ddH2O 14μl
  • digestion of mut(I+II+III)
    • digestion protocol
Total 20μl
DNA(mut(I+II)+III) 10μl
Digestion buffer 2μl
Enzyme 1 (X) 1μl
Enzyme 2 (P) 1μl
ddH2O 6μl
  • ligation: (1)eIF4A on pSB1C3 (2)mut(I+II)+III
    • ligation protocol
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1(eIF4A) 3μl
DNA2(pSB1C3) 2μl
ddH2O 3.5μl
NYMU 2010 9 16.png

2010.9.17

  • PCR of eIF4A(3 tubes)
    • PCR protocol
Total: 50μl X1.5μl FP RP Template length
Template 1.5μl negative
FP 1μl positive(mut(I+II)+III) eIF4A-split-A-FP eIF4A-split-B-RP mut(I+II)+III 1216
RP 1μl mut(I+II)+III eIF4A-FP eIF4A-split-B-RP mutI+mut(II+III) 1216
dNTP 2μl 3μl
10XBuff. 5μl 7.5μl
pfu 0.25μl 0.375μl
ddH2O 39.75μl 59.625μl
PCR Protocol
94 120s
94 30s
55 30s
72 80s 35 cycles
72 420s
  • PCR again
    • same protocol

2010.9.18

2010.9.20

  • purification of eIF4A(I+II)+III
  • ligation and transformation of eIF4A(I+II)+III and pSB1C3
    • ligation protocol
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1(eIF4A) 3μl eIF4A length 1216
DNA2(pSB1C3) 2μl pSB1C3 length 316
ddH2O 3.5μl


  • PCR of eIF4A-A,eIF4A-B(4tubes)
    • PCR protocol
Total: 50μl X2μl FP RP Template length
Template 1μl negative
FP 1μl positive(eIF4A) eIF4A-split-A-FP eIF4A-split-B-RP c212-2 1216
RP 1μl eIF4A-A eIF4A-split-A-FP eIF4A-split-A-RP mut(I+(II+III)) 1216
dNTP 2μl 4μl eIF4A-B eIF4A-split-B-FP eIF4A-split-B-RP mut(I+(II+III)) 1216
10XBuff. 5μl 10μl
pfu 0.25μl 0.5μl
ddH2O 39.75μl 79.5μl
PCR Protocol
94 120s
94 30s
55 30s
72 80s 35 cycles
72 420s

2010.9.21

  • PCR of GFP-A,GFP-B,RFP-A,RFP-B(6tubes)
    • PCR protocol
Total: 50μl X3μl FP RP Template length
Template 1μl negative
FP 1μl positive(RFP) VF2 VR RFP 776
RP 1μl GFP-A GFP-split-A-FP GFP-split-A-RP GFP 517
dNTP 2μl 6μl GFP-B GFP-split-B-FP GFP-split-B-RP GFP 298
10XBuff. 5μl 15μl RFP-B RFP-split-B-FP RFP-split-B-RP GFP 508
pfu 0.25μl 0.75μl RFP-B RFP-split-B-FP RFP-split-B-RP GFP 268
ddH2O 39.75μl 119.25μl
PCR Protocol
94 120s
94 30s
55 30s
72 80s 35 cycles
72 420s
  • 3 in 1 of eIF4A(I+II)+III and pSB1C3
  • PCR of eGFP(A)+eIF4A(A),eGFP(B)+eIF4A(B)
    • PCR protocol
Total: 50μl X2μl FP RP Template length
Template 1μl negative
FP 1μl positive VF2 VR eIF4A(eIF4A on pGEX) 1216
RP 1μl system GA GFP-split-A-FP eIF4A-split-A-RP GFP 1195
dNTP 2μl 4μl system GB GFP-split-B-FP eIF4A-split-B-RP GFP 847
10XBuff. 5μl 10μl system RA RFP-split-A-FP eIF4A-split-A-RP GFP 1181
pfu 0.25μl 0.5μl system RB RFP-split-A-FP eIF4A-split-A-RP GFP 818
ddH2O 39.75μl 79.5μl
PCR Protocol
94 120s
94 30s
55 30s
72 80s 35 cycles
72 420s
NYMU 2010 9 21.png
NYMU 2010 9 21 1.png

2010.9.22

  • gel extraction of system GA,system GB,system RA,system RB
  • digestion of eIF4A on pSB1C3,system GA,system GB, system RA,system RB
    • digestion protocol
Total 20μl
DNA 16μl
Digestion buffer 2μl
Enzyme 1 (X) 1μl
Enzyme 2 (P) 1μl
  • ligation of system GA,system GB,system RA
    • ligation protocol
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1(GA or GB) 0.5μl
DNA2(pSB1C3) 4.5μl
ddH2O 3.5μl
  • ligation of system RB
    • ligation protocol
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1(~) 1.5μl
DNA2(pSB1C3) 3.5μl
ddH2O 3.5μl
NYMU 2010 9 22.png
NYMU 2010 9 22 1.png

2010.9.23

  • digstion of pSB1C3+pLac(X,P)
    • digestion protocol
Total 20μl
DNA 16μl
Digestion buffer 2μl
Enzyme 1 (X) 1μl
Enzyme 2 (P) 1μl
  • gel extraction
  • ligation of system GA,system GB,system RA,system RB,
    • ligation protocol
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1(~) 2.5μl
DNA2(pSB1C3(X,P)) 5μl
ddH2O 1μl

2010.9.24

  • 3 in 1 of system GA, system GB, system RA, system RB
  • colony PCR of system GA, system GB, system RA, system RB
    • PCR protocol
Total: 50μl X5μl FP RP Template length
Template 1μl negative
FP 1μl positive(eIF4A on pGEX) eIF4A-split-A-Fp eIF4A-split-B-Rp eIF4A on pGEX 1216
RP 1μl system GA on pSB1C3 GFP-split-A-FP GFP-split-A-RP GFP 1195
dNTP 2μl 10μl system GB on pSB1C3 GFP-split-B-FP GFP-split-B-RP GFP 847
10XBuff. 5μl 25μl system RA on pSB1C3 RFP-split-B-FP RFP-split-B-RP GFP 1181
pfu 0.25μl 1.25μl system RB on pSB1C3 RFP-split-B-FP RFP-split-B-RP GFP 818
ddH2O 39.75μl 198.75μl
PCR Protocol
94 60s
94 15s
55 20s
72 80s 35 cycles
72 300s
NYMU 2010 9 24.png
NYMU 2010 9 24 1.png

2010.09.25

  • plasmid extration of

(1)GFP-eIF4A-A-1

(2)GFP-eIF4A-A-2(failed)

(3)GFP-eIF4A-B-1

(4)GFP-eIF4A-B-2(failed)

(5)RFP-eIF4A-A-1

(6)RFP-eIF4A-A-2

(7)RFP-eIF4A-B-1(failed)

(8)RFP-eIF4A-B-2

  • digestion of GA(XP),GB(XP),RA-1(XP),RA-2(XP),pGEX-e(P),pGEX-e(X)
    • digestion protocol
Total 20μl
DNA 16μl
Digestion buffer 2μl
Enzyme 1 (X) 1μl
Enzyme 2 (P) 1μl
  • ligation of GA,GB,RA,RB
    • ligation protocol of GA,GB
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1(pSB1A2) 2μl
DNA2(GA/GB)) 5μl
ddH2O 1.5μl
    • ligation protocol of RA,RB
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1(pSB1A2) 2μl
DNA2(RA/RB)) 1μl
ddH2O 5.5μl
  • digestion of pSB1A2(XP), pSB1C3(XP)
    • digestion protocol(X2)
Total 20μl
DNA 16μl
Digestion buffer 2μl
Enzyme 1 (X) 1μl
Enzyme 2 (P) 1μl
NYMU 2010 9 25.png
NYMU 2010 9 25 1.png

2010.09.26

  • colony PCR of GA(X2),GB(X2),RA(X2),RB(X2),on pSB1A2
    • PCR protocol
Total: 50μl X6μl length
Template 1μl negative
FP(VF2) 1μl positive(GFP2) 720+238
RP(VR) 1μl GFP-eIF4A-A-1,2 1387
dNTP 2μl 12μl GFP-eIF4A-B-1,2 1042
10XBuff. 5μl 30μl RFP-eIF4A-A-1,2 1387
tag 0.25μl 1.5μl RFP-eIF4A-B-1,2 1012
ddH2O 39.75μl 238.5μl
PCR Protocol
94 60s
94 15s
55 20s
72 90s 30 cycles
72 300s
  • digestion and gel extration of pSB1C3(XP), pSB1A2(XP)
NYMU 2010 9 26.png

2010.09.27

  • plasmid extraction of GFP-e-A-2,GFP-e-B-2,RFP-e-A-2,RFP-e-B-2
  • digestion of GFP-e-A-2,GFP-e-B-2,RFP-e-A-2,RFP-e-B-2
    • digestion protocol
Total 20μl
DNA 16μl
Digestion buffer 2μl
Enzyme 1 (X) 1μl
Enzyme 2 (P) 1μl
  • run gel and check.....failed!
  • ligation of GA,GB,RA,RB
    • ligation protocol of GA,GB
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1(pSB1A2) 2μl
DNA2(GA/GB)) 5μl
ddH2O 1.5μl
    • ligation protocol of RA,RB
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1(pSB1A2) 2μl
DNA2(RA/RB)) 1μl
ddH2O 5.5μl
  • transformation.
NYMU 2010 9 27.png
NYMU 2010 9 27 1.png
NYMU 2010 9 27 2.png

2010.09.28

  • 3 in 1 of colony PCR of GA,GB,RA,RB
    • PCR protocol
Total: 50μl X5μl length
Template 1μl negative
FP(VF2) 1μl positive(GFP2) 720+238
RP(VR) 1μl GFP-eIF4A-A-1,2 1387
dNTP 2μl 10μl GFP-eIF4A-B-1,2 1042
10XBuff. 5μl 25μl RFP-eIF4A-A-1,2 1387
tag 0.25μl 1.25μl RFP-eIF4A-B-1,2 1012
ddH2O 39.75μl 198.75μl
PCR Protocol
94 60s
94 15s
55 20s
72 90s 30 cycles
72 300s
NYMU 2010 9 28.png

2010.09.29

  • plasmid extractio of GA(1),GB(2),RA(1),RB(2)
  • digestion of GA(1),GB(2),RA(1),RB(2)
    • digestion protocol
Total 20μl
DNA 16μl
Digestion buffer 2μl
Enzyme 1 (X) 1μl
Enzyme 2 (P) 1μl

2010.09.30

  • ligation of GA,GB,RA,RB on pSB1A2
    • ligation protocol of GA,GB
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1(pSB1A2) 2μl
DNA2(GA/GB)) 5μl
ddH2O 1.5μl
    • ligation protocol of RA,RB
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1(pSB1A2) 2μl
DNA2(RA/RB)) 1μl
ddH2O 5.5μl

2010.10.01

  • 3in1 of GA(2),GB(2),RA(1),J13002(2) (use liquid to replace for the LB buffer)
    • PCR protocol
Total: 50μl X4.5μl
Template 1μl negative
FP 1μl positive(GFP2)
RP 1μl
dNTP 2μl 9μl
10XBuff. 5μl 22.5μl
tag 0.25μl 1.125μl
ddH2O 39.75μl 178.875μl
PCR Protocol
94 60s
94 15s
55 20s
72 80s 35 cycles
72 300s
  • digestion GeA,GeB,ReA,ReB
    • digestion protocol
Total 20μl
DNA 16μl
Digestion buffer 2μl
Enzyme 1 (X) 1μl
Enzyme 2 (P) 1μl
  • PCR of GeA,GeB,ReA,ReB
    • PCR protocol
Total: 50μl X4μl FP RP Template length
Template 1μl negative
FP 1μl
RP 1μl system GA GFP-split-A-FP eIF4A-split-A-RP
dNTP 2μl 8μl system GB GFP-split-B-FP eIF4A-split-B-RP
10XBuff. 5μl 20μl system RA RFP-split-A-FP eIF4A-split-A-RP
pfu 0.25μl 1μl system RB RFP-split-B-FP eIF4A-split-B-RP
ddH2O 39.75μl 159μl
PCR Protocol
94 120s
94 30s
55 30s
72 80s 35 cycles
72 420s
NYMU 2010 10 01.png

2010.10.02

  • plasmid extraction of GA, GB, RA, J13002
  • digestion(XP) to check length
  • purify the PCR yesterday and digest(DNA:16)
  • gel extraciton
  • GA.RB digest again.
  • RUSULT:
    • THe result of digestion from plasmid extraction is wrong.
    • The reusult of PCR is right

2010.10.03

  • colony PCR of GeA, GeB, ReA, ReB on J13002
    • PCR protocol
Total: 50μl X10μl
Template 1μl
FP 1μl positive(GFP2)
RP 1μl system GA on J13002
dNTP 2μl 20μl system GB on J13002
10XBuff. 5μl 50μl system RA on J13002
pfu 0.25μl 2.5μl system RB on J13002
ddH2O 39.75μl 397.5μl
PCR Protocol
94 60s
94 15s
55 20s
72 100s 30 cycles
72 300s
  • ligation of aptamer(XP)+pLac(SP)
    • ligation protocol
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1(pLac) 1μl
DNA2(aptamer) 3μl
ddH2O 4.5μl
  • digestion of J13002(SP)
    • digestion protocol
Total 20μl
DNA 10μl
Digestion buffer 2μl
Enzyme 1 (S) 1μl
Enzyme 2 (P) 1μl
ddH2O 6μl

2010.10.04

  • purification of J13002(SP)
  • 3in1 of aptamer(XP)+pLAC(SP)
    • colony PCR
      • PCR protocol
Total: 50μl X2μl
Template 1μl negative
FP 1μl positive(GFP2)
RP 1μl
dNTP 2μl 4μl
10XBuff. 5μl 10μl
tag 0.25μl 0.5μl
ddH2O 39.75μl 79.5μl
PCR Protocol
94 60s
94 15s
55 20s
72 40s 30 cycles
72 300s
  • ligation of J13002(SP)+GA,GB,RA,RB(XP)
    • ligation protocol of GA,GB
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1(J13002) 4μl
DNA2(~) 1μl
ddH2O 3.5μl
    • ligation protocol of RA,RB
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1(J13002) 4.5μl
DNA2(~) 0.5μl
ddH2O 3.5μl
  • transformation

2010.10.06

  • 3in1 of pTet+RBS+GA,GB,RA,RB
    • colony PCR
    • PCR protocol
Total: 50μl X6μl
Template 1μl
FP 1μl
RP 1μl
dNTP 2μl 12μl
10XBuff. 5μl 30μl
pfu 0.25μl 1.5μl
ddH2O 39.75μl 238.5μl
PCR Protocol
94 60s
94 15s
55 20s
72 100s 30 cycles
72 300s
  • plasmid extraction of aptamer(XP)+pLac(SP)]
  • run gel of pTet+RBS+GA(X5),GB(X4),RA(X4),RB(X4)
  • cultivate LB: GA tube5, GB tube3, RA tube1, RA tube2

2010.10.07

  • plasmid extraction of pTet+RBS+GA,GB,RA1,RA2
  • digestion of pTet+RBS+GA,GB,RA1,RA2
    • digestion protocol
Total 20μl
DNA 16μl
Digestion buffer 2μl
Enzyme 1 (X) 1μl
Enzyme 2 (P) 1μl
  • digestion of J13002(SP) > purification > ligation of J13002(SP)+RBS(XP)

2010.10.08

  • purification of pLac+apt(SP)
  • digestion of pSB1C3(XP),pLac+apt(XP)
    • digestion protocol
Total 20μl
DNA 16μl
Digestion buffer 2μl
Enzyme 1 (X) 1μl
Enzyme 2 (P) 1μl
  • run gel of pSB1C3(XP),pLac+apt(XP),terminator(XP) then gel extraction.
  • PCR of J13002+GB,J13002+RB1,J13002+RB2
    • PCR protocol
Total: 50μl X2μl
Template 1μl
FP 1μl
RP 1μl
dNTP 2μl 4μl
10XBuff. 5μl 10μl
tag 0.25μl 0.5μl
ddH2O 39.75μl 79.5μl
PCR Protocol
94 60s
94 15s
55 20s
72 100s 30 cycles
72 300s
  • 3in1 of J13002+GB,J13002+RB1,J13002+RB2

2010.10.09

  • PCR of J13002+GB,J13002+RB1,J13002+RB2
    • colony PCR protocol
Total: 50μl X2μl
Template 1μl
FP 1μl
RP 1μl
dNTP 2μl 4μl
10XBuff. 5μl 10μl
tag 0.25μl 0.5μl
ddH2O 39.75μl 79.5μl
PCR Protocol
94 60s
94 15s
55 20s
72 100s 30 cycles
72 300s
  • plasmid extraction of J13002+GB,J13002+RB1,J13002+RB2
  • digestion of J13002+GB,J13002+RB1,J13002+RB2
    • digestion protocol
Total 20μl
DNA 16μl
Digestion buffer 2μl
Enzyme 1 (X) 1μl
Enzyme 2 (P) 1μl
    • run gel to check >> all failed.
  • purification of

(1)pLac+aptamer(XP)

(2)terminator(XP)

(3)pSB1C3(XP)

  • ligation of

(1)eIF4A+pSB1C3

(2)pLac+aptamer(SP)+terminator(XP)

(3)J13002(SP)+GA/GB/RA/RB

    • ligation protocol of GA,GB
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1(pSB1A2) 2μl
DNA2(GA/GB)) 5μl
ddH2O 1.5μl
    • ligation protocol of RA,RB
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1(pSB1A2) 2μl
DNA2(GA/GB)) 5μl
ddH2O 1.5μl

2010.10.10

  • colony PCR of pSB1C3+eIF4A,pSB1C3+pLac+aptamer,J13002+ReB,J13002+GeB
    • colony PCR protocol
Total: 50μl X2.5μl
Template 1μl
FP(VF2) 1μl 2.5μl
RP(VR) 1μl 2.5μl
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 100s 30 cycles
72 300s
  • digestion of J13002
    • digestion protocol
Total 20μl
DNA 8μl
Digestion buffer 2μl
Enzyme 1 (S) 1μl
Enzyme 2 (P) 1μl
ddH2O 8μl
  • ligation of pSB1C3+eIF4A
    • ligaton protocol
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1(pSB1C3) 2μl
DNA2(eIF4A) 3μl
ddH2O 3.5μl
  • ligation of pSB1C3+pLac+aptamer,J13002+GeA,J13002+GeB
    • ligation protocol
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1(pSB1C3/J13002) 1.5μl
DNA2(pLac+aptamer/GeA,GeB) 3.5μl
ddH2O 3.5μl
  • ligation ofJ13002+ReA,J13002+ReB
    • ligation protocol
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1(J13002) 2μl
DNA2(ReA/ReB) 3μl
ddH2O 3.5μl

2010.10.11

  • 3in1 of pSB1C3+GeA,pSB1C3+GeB,pSB1C3+ReA,pSB1C3+ReB,pSB1C3+pLac+aptamer
    • colony PCR protocol
Total: 50μl X10μl
Template 1μl
FP 1μl
RP 1μl
dNTP 2μl 20μl
10XBuff. 5μl 50μl
tag 0.25μl 2.5μl
ddH2O 39.75μl 397.5μl
PCR Protocol
94 60s
94 15s
55 20s
72 100s 30 cycles
72 300s

2010.10.12

  • digstion of J13002+GeA,J13002+ReA(x2),J13002+ReB(x2),pSB1C3+pLac+aptamer(x2)
    • digestion protocol
Total 20μl
DNA 8μl
Digestion buffer 2μl
Enzyme 1 (S) 1μl
Enzyme 2 (P) 1μl
ddH2O 8μl

2010.10.13

  • digestion of J13002+GeA,J13002+ReA(X,P),J13002+ReB(x2),pSB1C3+pLac(S,P)→check length
    • digestion protocol
Total 20μl
DNA 16μl
Digestion buffer 2μl
Enzyme 1 (X) 1μl
Enzyme 2 (P) 1μl
  • 3in1 of C3+GA(X1),C3+GB(X3), C3+eIF4A(X5)
    • colony PCR protocol
Total: 50μl X6μl
Template 1μl
FP 1μl
RP 1μl
dNTP 2μl 12μl
10XBuff. 5μl 30μl
tag 0.25μl 1.5μl
ddH2O 39.75μl 238.5μl
PCR Protocol
94 60s
94 15s
55 20s
72 100s 30 cycles
72 300s
  • run gel of JRA(X5),JRB(X2)
  • ligation of

(1)J13002+GA,GB,RA,GB

(2)C3+GA,GB,RA,GB

(3)C3+pLac+aptamer+terminator

    • ligation protocol of GA,GB
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1(pSB1A2) 2μl
DNA2(GA/GB)) 5μl
ddH2O 1.5μl

2010.10.14

  • 3in1 of J13002+GA,GB,RA,RB (all failed)
    • colony PCR protocol
Total: 50μl X5μl
Template 1μl
FP 1μl
RP 1μl
dNTP 2μl 10μl
10XBuff. 5μl 25μl
tag 0.25μl 1.25μl
ddH2O 39.75μl 198.75μl
PCR Protocol
94 60s
94 15s
55 20s
72 100s 30 cycles
72 300s
  • real PCR of GeA,GeB,ReA,ReB
Total: 50μl X5μl FP RP
Template 1μl negative
FP 1μl positive(eIF4A) eIF4A-Fp eIF4A-split-B-Rp
RP 1μl GeA GFP-split-A-FP eIF4A-split-A-Rp
dNTP 2μl 10μl GeB GFP-split-B-FP eIF4A-split-B-Rp
10XBuff. 5μl 25μl ReA RFP-split-A-FP eIF4A-split-A-Rp
pfu 0.25μl 1.25μl ReB RFP-split-B-FP eIF4A-split-B-Rp
ddH2O 39.75μl 198.75μl
PCR Protocol
94 120s
94 30s
55 30s
72 100s 35 cycles
72 600s
  • digestion of eIF4A(for biobrick)
    • digestion protocol
Total 20μl
DNA 16μl
Digestion buffer 2μl
Enzyme 1 (X) 1μl
Enzyme 2 (P) 1μl
  • digestion of pLac+aptamer(SP)
    • digestion protocol
Total 20μl
DNA 16μl
Digestion buffer 2μl
Enzyme 1 (S) 1μl
Enzyme 2 (P) 1μl
  • digestion of GeA,GeB,ReA,ReB,eIF4A(all from PCR, biobrick cutting site)
    • digestion protocol
Total 20μl
DNA 16μl
Digestion buffer 2μl
Enzyme 1 (X) 1μl
Enzyme 2 (P) 1μl
  • tag PCR of negative > to check if there has contamination
Total: 50μl X2μl FP RP
Template 1μl negative
FP 1μl positive(eIF4A) eIF4A-Fp eIF4A-split-B-Rp
RP 1μl GeA GFP-split-A-FP eIF4A-split-A-Rp
dNTP 2μl 4μl GeB GFP-split-B-FP eIF4A-split-B-Rp
10XBuff. 5μl 10μl ReA RFP-split-A-FP eIF4A-split-A-Rp
tag 0.25μl 0.5μl ReB RFP-split-B-FP eIF4A-split-B-Rp
ddH2O 39.75μl 79.5μl
PCR Protocol
94 120s
94 30s
55 30s
72 100s 35 cycles
72 600s

2010.10.15

  • ligation of pSB1C3+GeA/GeB/ReA/ReB/eIF4A/pLac+eIF4A+treminator,pSB1A2+J13002+GeA/GeB/ReA/ReB
    • ligation protocol
    • ligation protocol of RA,RB
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1() 4.5μl
DNA2(~) 0.5μl
ddH2O 3.5μl
  • digestion of pSB1C3
    • digestion protocol
Total 20μl
DNA 16μl
Digestion buffer 2μl
Enzyme 1 (X) 1μl
Enzyme 2 (P) 1μl
  • PCR of GeA/ReA
    • PCR protocol
Total: 50μl X2μl FP RP
Template 1μl negative
FP 1μl positive(eIF4A) eIF4A-Fp eIF4A-split-B-Rp
RP 1μl GeA GFP-split-A-FP eIF4A-split-A-Rp
dNTP(KOD) 4μl 8μl GeB GFP-split-B-FP eIF4A-split-B-Rp
10XBuffer(KOD) 5μl 10μl ReA RFP-split-A-FP eIF4A-split-A-Rp
KOD 1μl 2μl
ddH2O 33μl 198.75μl
MgSO4 4μl 8μl
PCR Protocol
94 120s
94 30s
55 30s
72 100s 35 cycles
72 600s


  • 3in1 of J13002+GB,pSB1C3+pLac+aptamer+terminator
    • colony PCR protocol
Total: 50μl X3μl
Template 1μl
FP 1μl 3μl
RP 1μl 3μl
dNTP 2μl 6μl
10XBuff. 5μl 15μl
tag 0.25μl 0.75μl
ddH2O 39.75μl 119.75μl
PCR Protocol
94 60s
94 15s
55 20s
72 100s 30 cycles
72 300s

2010.10.16

  • ligation of pSB1C3+GeA/GeB/ReA/ReB/eIF4A/pLac+eIF4A+treminator,pSB1A2+J13002+GeA/GeB/ReA/ReB
    • ligation protocol
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1(J13002) 4.5μl
DNA2(~) 0.5μl
ddH2O 3.5μl
  • digestion of C3+GeA(X,P)/GeB(X,P)/ReA(X,P),J13002+GeB(S,P)
    • digestion protocol
Total 20μl
DNA 16μl
Digestion buffer 2μl
Enzyme 1 (X,S) 1μl
Enzyme 2 (P) 1μl

2010.10.17

  • 3in1 of J13002+ReA/GeA/ReB/GeB+terminator
    • colony PCR protocol
Total: 50μl X3.6μl
Template 1μl
FP 1μl 3.6μl
RP 1μl 3.6μl
dNTP 2μl 7.2μl
10XBuff. 5μl 18μl
tag 0.25μl 0.9μl
ddH2O 39.75μl 141.3μl
PCR Protocol
94 60s
94 15s
55 20s
72 100s 30 cycles
72 300s
  • ligation of pSB1C3+GeB/eIF4A,pSB1A2+J13002+GeB
    • ligation protocol
    • ligation protocol of RA,RB
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1(J13002) 1μl
DNA2(~) 2μl
ddH2O 5.5μl

2010.10.18

  • 3in1 of JGeA, JReA, JReB, C3+ReB
    • colony PCR protocol
Total: 50μl X6μl
Template 1μl
FP 1μl
RP 1μl
dNTP 2μl 12μl
10XBuff. 5μl 30μl
tag 0.25μl 1.5μl
ddH2O 39.75μl 238.5μl
PCR Protocol
94 60s
94 15s
55 20s
72 100s 30 cycles
72 300s

2010.10.19

  • ligation of JReA, JReB, C3+GeA,GeB,ReA,eIF4A
    • ligation protocol
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1(vector) 1μl
DNA2(~)) 2μl
ddH2O 5.5μl
  • digestion of JGeA
    • digestion protocol
Total 20μl
DNA 10μl
Digestion buffer 2μl
Enzyme 1 (S) 1μl
Enzyme 2 (P) 1μl
ddH2O 6μl

2010.10.20

  • 3in1 of

(1)JGeA+terminator

(2)J13002+ReA(old)

(3)J13002+ReA(new)

    • colony PCR protocol
Total: 50μl X2μl
Template 1μl
FP 1μl
RP 1μl
dNTP 2μl 4μl
10XBuff. 5μl 10μl
tag 0.25μl 0.5μl
ddH2O 39.75μl 79.5μl
PCR Protocol
94 60s
94 15s
55 20s
72 100s 30 cycles
72 300s
  • ligation of

(1)JGeA+terminator

(2)JReA

(3)JReB

(4)C3+GeA

(5)C3+GeB

(6)C3+ReA

(7)C3+ReB

(8)C3+eIF4A

    • ligation protocol of GA,GB
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1(vector) 1μl
DNA2(~) 2μl
ddH2O 5.5μl

2010.10.21

  • ligation of J13002 ReB,pSB1C3+ReA/ReB/GeA/GeB/eIF4A
    • ligation protocol
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1(vector) 1μl
DNA2(~) 2μl
ddH2O 5.5μl

2010.10.22

  • ligation of J13002+ReA/ReB,pSB1C3+eIF4A/GeA/GeB/ReA/ReB
    • ligation protocol
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1(vector) 1μl
DNA2(~) 2μl
ddH2O 5.5μl

2010.10.23

  • 3in1 of JGeA, JReA, JReB
    • colony PCR protocol
Total: 50μl X5μl
Template 1μl
FP 1μl
RP 1μl
dNTP 2μl 10μl
10XBuff. 5μl 25μl
tag 0.25μl 1.25μl
ddH2O 39.75μl 198.75μl
PCR Protocol
94 60s
94 15s
55 20s
72 100s 30 cycles
72 300s

2010.10.24

  • 3in1 of JReA, JReB, C3+ReB/eIF4A/GA/GB/RA
    • colony PCR protocol
Total: 50μl X6.6μl
Template 1μl
FP 1μl
RP 1μl
dNTP 2μl 13.2μl
10XBuff. 5μl 33μl
tag 0.25μl 1.65μl
ddH2O 39.75μl 262.35μl
PCR Protocol
94 60s
94 15s
55 20s
72 100s 30 cycles
72 300s
DSC05773.JPG
  • digestion of JGeAT,JGeT
    • digestion protocol
Total 20μl
DNA 16μl
Digestion buffer 2μl
Enzyme 1 (X) 1μl
Enzyme 2 (P) 1μl

2010.10.25

2010.10.26

  • ligation of pSB1C3+eIF4A/GeA/ReA/ReB
    • ligation protocol
Total 10μl
buffer 1μl
ligase 0.5μl
DNA1(vector) 1μl
DNA2(~) 2μl
ddH2O 5.5μl
  • 3in1 of pSB1C3+GeA/ReA/ReB


Retrieved from "http://2010.igem.org/MRNA_Binding_Experiments"