Team:Newcastle/5 August 2010

From 2010.igem.org

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==Aim==
==Aim==
-
The aim of the experiment is to perform gel electrophoresis for all the 6 PCR reactions which took place yesterday 4th August, 2010 and thus confirm that all 6 PCR reactions were successful.
+
The aim of the experiment is to check for the PCR amplified RocF frangments that was performed on 4th August, 2010 by using gel electrophorsis.
==Materials and Protocol==
==Materials and Protocol==
Line 12: Line 12:
[[Image:Newcastle_050810_first_PCR_gel.png|400px]]
[[Image:Newcastle_050810_first_PCR_gel.png|400px]]
-
Figure 1: Gel electrophoresis of the pSB1C3, Pspac_oid promoter, ''rocF'' fragments and double terminator.
+
'''Figure 1''': Gel electrophoresis of the pSB1C3, Pspac_oid promoter, ''rocF'' fragments and double terminator.
-
* '''Lane 1''': 1kb DNA ladder
+
* '''Lane 1''': 1 Kb DNA ladder
* '''Lane 2''': BioBrick compatible vector pSB1C3
* '''Lane 2''': BioBrick compatible vector pSB1C3
* '''Lane 3''': Pspac_oid promoter
* '''Lane 3''': Pspac_oid promoter
Line 21: Line 21:
* '''Lane 6''': 3rd fragment of ''rocF'' CDS
* '''Lane 6''': 3rd fragment of ''rocF'' CDS
* '''Lane 7''': Double Terminator
* '''Lane 7''': Double Terminator
-
* '''Lane 8''': 1kb DNA ladder
+
* '''Lane 8''': 1 Kb DNA ladder
{|border=1
{|border=1
Line 35: Line 35:
|'''Size of the Fragment (in bp)'''
|'''Size of the Fragment (in bp)'''
|2072 approx.
|2072 approx.
-
|106 approx.
+
|148 approx.
|246 approx.
|246 approx.
|597 approx.
|597 approx.
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==Discussion==
==Discussion==
-
We found bands in the lanes 2,4,5,6 and 7 of the correct sizes but lane 3 did not contain any band.
+
Correct sized bands were observed in lanes 2,4,5,6 and 7. However lane 3 did not contain any band.
==Conclusion==
==Conclusion==
-
This experiment shows that the PCR reaction was successful for all the fragments apart from Pspac_oid promoter which was present in the lane 3 and did not show any band. This could be because of the following problems:
+
PCR reaction was successful for all the fragments apart from Pspac_oid promoter which was represented in lane 3. This could be due to the following prpeblems:
# Primer sequences could be incorrect.
# Primer sequences could be incorrect.
# Melting temperature could be incorrect.
# Melting temperature could be incorrect.
Line 54: Line 54:
==Solution for the problem==
==Solution for the problem==
# Check the primer sequences so as to eliminate any problems associated with the primer sequence.
# Check the primer sequences so as to eliminate any problems associated with the primer sequence.
-
# Perform PCR reactions for the Pspac_oid fragment with 3 different melting temperatures viz. 50°C, 51°C and 52°C.
+
# Perform PCR reactions for the Pspac_oid fragment with 3 different melting temperatures at 50°C, 51°C and 52°C.
-
 
+
=Amplification of the Pspac_oid promoter and RocF fragments by PCR=
-
=Amplification of Pspac_oid promoter by PCR=
+
==Aim==
==Aim==
-
The aim of this experiment is to amplify Pspac_oid promoter fragment from plasmid pMutin4 for the construction of [[Team:Newcastle/Urease|''rocF'' BioBrick]] with the help of 3 different Phusion PCR.
+
The aim of this experiment is to amplify the Pspac_oid promoter fragment from the plasmid pMutin4 for the construction of the [[Team:Newcastle/Urease|''rocF'' BioBrick]] using 3 different melting temperatures in the Phusion PCR protocol, as well as to rerun the gel electrophoresis of the RocF fragments and the double terminator fragments obtained this morning.
==Materials and Protocol==
==Materials and Protocol==
-
Please refer to  [[Team:Newcastle/PCR| PCR]] for Phusion PCR protocol. The details for the 3 PCR reactions are mentioned below:
+
Please refer to  [[Team:Newcastle/PCR| PCR]] for the Phusion PCR protocol. The details for the 3 PCR reactions are mentioned below:
===PCR===
===PCR===
Line 106: Line 105:
'''Table 2''': Table represents 3 different Phusion PCR reactions for the amplification of Pspac_oid promoter, so that it can be ligated together with other fragments for the construction of ''rocF'' with the help of Gibson Cloning method.
'''Table 2''': Table represents 3 different Phusion PCR reactions for the amplification of Pspac_oid promoter, so that it can be ligated together with other fragments for the construction of ''rocF'' with the help of Gibson Cloning method.
-
* The extension rate of the Phusion polymerase is 1Kb/ 30 seconds. Thus the extension time of each and every PCR reaction is slightly different.
+
* The extension rate of the Phusion polymerase is 1Kb/ 30 seconds. Therefore the extension time of each PCR reaction is different.
-
* For learning about the ''rocF'' fragments, please refer to the [[Media:Cloning_strategy_for_rocF.pdf|Cloning strategy for ''rocF'']].
+
* To learn more about the ''rocF'' fragments, please refer to the [[Media:Cloning_strategy_for_rocF.pdf|Cloning strategy for ''rocF'']].
-
 
+
-
==Discussion==
+
-
All the 3 Phusion PCR reactions were done however, gel electrophoresis will be done later today, to check whether the fragments have actually amplified or not.
+
-
 
+
-
==Conclusion==
+
-
Today afternoon, we would be running gel electrophoresis to check the outcome of the 3 PCR reactions and later all the fragments will be ligated with help of Gibson protocol.
+
-
 
+
-
=Concrete Tensile Splitting Test=
+
-
 
+
-
==Aim==
+
-
 
+
-
To get samples of cracked concrete for Bacilla Filla to fill up the cracks and to determine the tensile strength of concrete before the cracks are filled up.
+
-
 
+
-
==Materials==
+
-
 
+
-
* Concrete cylinder
+
-
* Jubilee clips
+
-
 
+
-
==Procedure==
+
-
 
+
-
# A concrete cylinder was made beforehand and left for more than 28 days to cure so that a straight line of crack will form down the diameter of the cylinder.
+
-
# The cylinder is placed on two diametrically opposed loading generators. Two pieces of plywood are placed between the loading plates and the concrete cylinder to prevent failure in compression.
+
-
# The generator is then started for loading until the cylinder forms a crack down the diameter. The maximum load is recorded and tensile strength of the concrete cylinder is calculated.
+
-
 
+
-
{|
+
-
|-
+
-
|[[Image:Newcastle_Concrete_6.jpg|thumb|Concrete cylinder]]
+
-
|[[Image:Newcastle_Concrete_7.jpg|thumb|Loading generator]]
+
-
|[[Image:Newcastle_Hoppings_003.JPG|thumb|Philamentous!!!]]
+
-
|[[Image:Newcastle_Hoppings_004.JPG|thumb]]
+
-
|-
+
-
[[Image:Newcastle_Concrete_6.jpg|275px|left|Concrete cylinder]]
+
==Result==
==Result==
-
The maximum load that is recorded from the test is 171.3kN.  
+
[[Image:Newcastle_050810_PCR_100bp_second_gel.png|500px]]
-
==Discussion==
+
'''Figure 2''': Gel electrophoresis of the pSB1C3, Pspac_oid promoter, rocF fragments and double terminator.
-
From the formula f=(2P)/(πBD),  
+
-
where f=tensile strength,
+
-
P=Maximum applied load,
+
-
B=Depth of cylinder,
+
-
D=Diameter of cylinder,
+
-
We calculate that the maximum tensile strength of this concrete cylinder is  .
 
-
 
-
==Conclusion==
 
-
We now have the original tensile strength of the concrete cylinder, which is  . We will test its tensile strength again after the concrete has been filled up by Bacilla Filla.
 
-
 
-
=Gel Electrophoresis for the Amplified Fragments of ''rocF''=
 
-
 
-
==Aim==
 
-
The aim of the experiment is to perform gel electrophoresis for Pspac_oid PCR reaction products which took place today and on 3 fragments of ''rocF'' CDS and Double terminatorand PCR products thus confirming that all the PCR reactions were successful.
 
-
 
-
==Materials and Protocol==
 
-
Please refer to: [[Team:Newcastle/Gel electrophoresis| Gel electrophoresis]].
 
-
 
-
==Result==
 
-
 
-
[[Image:Newcastle 050810 PCR 100bp ladder.jpg|500px]]
 
* '''Lane 1''': 100bp DNA ladder
* '''Lane 1''': 100bp DNA ladder
* '''Lane 2''': 1st fragment of ''rocF'' CDS
* '''Lane 2''': 1st fragment of ''rocF'' CDS
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* '''Lane 8''': Pspac_oid promoter (Tm 52°C i.e. Tube 3 of PCR reaction which is mentioned above)
* '''Lane 8''': Pspac_oid promoter (Tm 52°C i.e. Tube 3 of PCR reaction which is mentioned above)
* '''Lane 9''': 100bp DNA ladder
* '''Lane 9''': 100bp DNA ladder
 +
{|border=1
{|border=1
Line 187: Line 134:
|-
|-
|'''Size of the Fragment (in bp)'''
|'''Size of the Fragment (in bp)'''
-
|106 approx.
+
|148 approx.
|246 approx.
|246 approx.
|597 approx.
|597 approx.
Line 193: Line 140:
|116 approx.
|116 approx.
|}
|}
 +
'''Table 3''': Table represents the size of the fragments represented as bands on the gel in their corresponding lanes.
'''Table 3''': Table represents the size of the fragments represented as bands on the gel in their corresponding lanes.
==Discussion==
==Discussion==
-
We found bands in the lanes 2,3,4 and 5 of the correct sizes but lanes 6,7, and 8 did not contain any band. When the gel electrophoresis was performed in the morning, similar results were found on the gel. We checked the coding sequence of the primers and they were correct and we also used 3 different melting temperatures for the PCR reaction so as to eliminate any chances of having a calculation mistake for melting temperature.
+
Correct bands size was observed in all lanes. The three different melting temperature used during the PCR for the Pspac_oid promoter were successful.
==Conclusion==
==Conclusion==
-
This experiment shows that the PCR reaction was successful for all the fragments apart from Pspac_oid promoter which was present in the lanes 6,7, and 8 and did not show any band. This could be because of the following problems:
+
The amplified fragments of ''RocF'', double terminator and the Pspac_oid promoter have been successful.
-
# Plasmid pMutin4 could have degenerated due to long term storage.
+
-
==Solution for the problem==
+
=Concrete Tensile Splitting Test=
-
# Use a different stock of the plasmid pMutin4 and perform PCR reaction for the amplification of Pspac_oid promoter.
+
 +
==Aim==
-
=Amplification of Pspac_oid promoter and ''lacI'' from a different stock of pMutin4 by PCR=
+
To obtain samples of cracked concrete for BacillaFilla to fill up the cracks and also to determine the tensile strength of concrete before the cracks are filled up.
-
==Aim==
+
==Materials==
-
The aim of this experiment is to amplify Pspac_oid promoter fragment and ''lacI'' fragmentfrom a different stock of plasmid pMutin4 with the help of 2 different Phusion PCR.
+
-
==Materials and Protocol==
+
* Concrete cylinder
-
Please refer to  [[Team:Newcastle/PCR| PCR]] for Phusion PCR protocol. The details for the 3 PCR reactions are mentioned below:
+
* Jubilee clips
-
===PCR===
+
==Procedure==
-
{|border=1
+
 
 +
# A concrete cylinder was made beforehand and left for more than 28 days to cure so that a straight line of crack will form down the diameter of the cylinder.
 +
# The cylinder is placed on two diametrically opposed loading generators. Two pieces of plywood are placed between the loading plates and the concrete cylinder to prevent failure in compression.
 +
# The generator is then started for loading until the cylinder forms a crack down the diameter. The maximum load is recorded and tensile strength of the concrete cylinder is calculated.
 +
 
 +
{|
|-
|-
-
!'''Tube'''
+
|[[Image:Newcastle_Concrete_6.jpg|thumb|Concrete cylinder]]
-
!'''Part to be amplified'''
+
|[[Image:Newcastle_Concrete_7.jpg|thumb|Loading generator]]
-
!'''DNA fragment consisting the part'''
+
|[[Image:Newcastle_Concrete_8.jpg|thumb|Cracked concrete]]
-
!'''Forward primer'''
+
|[[Image:Newcastle_Concrete_9.jpg|thumb|Steven giving his baby to Phil]]
-
!'''Reverse Primer'''
+
-
!'''Melting Temperature (Tm in °C) '''
+
-
!'''Size of the fragment (in bp)'''
+
-
!'''Extension time* (in seconds)'''
+
-
|-
+
-
|1
+
-
|''LacI''
+
-
|pMutin4
+
-
|P1P1 forward
+
-
|P2P1 reverse
+
-
|54
+
-
|1100 approx.
+
-
|15
+
-
|-
+
-
|2
+
-
|Pspacoid Promoter
+
-
|pMutin4
+
-
|P1P1 forward
+
-
|P2P1 reverse
+
-
|50
+
-
|106 approx.
+
-
|15
+
|}
|}
-
'''Table 4''': Table represents 3 different Phusion PCR reactions for the amplification of Pspac_oid promoter from a different source of plasmid pMutin4, so that it can be ligated together with other fragments for the construction of ''rocF'' with the help of Gibson Cloning method.
+
[[Image:Newcastle Concrete 10.jpg|thumb|Failure load]]
-
* The extension rate of the Phusion polymerase is 1Kb/ 30 seconds. Thus the extension time of each and every PCR reaction is slightly different.
+
 
 +
==Result==
 +
 
 +
The maximum load that is recorded from the test is 171.3 kN.
==Discussion==
==Discussion==
-
Both Phusion PCR reactions were done however, gel electrophoresis was done on 6th August, 2010, to check whether the fragments have actually amplified or not. In this experiment, ''lacI'' is acting as control to check whether the new stock of the plasmid pMutin4 is perfect or not.
+
From the formula f=(2P)/(πBD),  
 +
where f=tensile strength,
 +
P=Maximum applied load,
 +
B=Depth of cylinder,
 +
D=Diameter of cylinder,
 +
 
 +
With the depth and diameter of the cylinder as 30m and 15m respectively, we calculated the maximum tensile strength of this concrete cylinder to be 242.3 kN/m².
==Conclusion==
==Conclusion==
-
Tomorrow, we would be running gel electrophoresis to check the outcome of both the PCR reactions and later all the fragments related to the construction of ''rocF'' BioBrick will be ligated with help of Gibson protocol.
+
We now have the original tensile strength of the concrete cylinder, which is 242.3 kN/m². We will test its tensile strength again after the concrete has been filled up by BacillaFilla.
-
{{Team:Newcastle/footer}}
+

Latest revision as of 22:57, 27 October 2010

iGEM Homepage Newcastle University BacillaFilla Homepage Image Map

Contents

Gel Electrophoresis for the Amplified Fragments of rocF

Aim

The aim of the experiment is to check for the PCR amplified RocF frangments that was performed on 4th August, 2010 by using gel electrophorsis.

Materials and Protocol

Please refer to: Gel electrophoresis.

Result

Newcastle 050810 first PCR gel.png

Figure 1: Gel electrophoresis of the pSB1C3, Pspac_oid promoter, rocF fragments and double terminator.

  • Lane 1: 1 Kb DNA ladder
  • Lane 2: BioBrick compatible vector pSB1C3
  • Lane 3: Pspac_oid promoter
  • Lane 4: 1st fragment of rocF CDS
  • Lane 5: 2nd fragment of rocF CDS
  • Lane 6: 3rd fragment of rocF CDS
  • Lane 7: Double Terminator
  • Lane 8: 1 Kb DNA ladder
Biobrick compatible vector pSB1C3 Pspac_oid pormoter 1st fragment of rocF CDS 2nd fragment of rocF CDS) 3rd fragment of rocF CDS Double Terminator
Size of the Fragment (in bp) 2072 approx. 148 approx. 246 approx. 597 approx. 125 approx. 116 approx.

Table 1: Table represents the size of the fragments represented as bands on the gel in their corresponding lanes.

Discussion

Correct sized bands were observed in lanes 2,4,5,6 and 7. However lane 3 did not contain any band.

Conclusion

PCR reaction was successful for all the fragments apart from Pspac_oid promoter which was represented in lane 3. This could be due to the following prpeblems:

  1. Primer sequences could be incorrect.
  2. Melting temperature could be incorrect.
  3. Plasmid pMutin4 could have degenerated due to long term storage.

Solution for the problem

  1. Check the primer sequences so as to eliminate any problems associated with the primer sequence.
  2. Perform PCR reactions for the Pspac_oid fragment with 3 different melting temperatures at 50°C, 51°C and 52°C.

Amplification of the Pspac_oid promoter and RocF fragments by PCR

Aim

The aim of this experiment is to amplify the Pspac_oid promoter fragment from the plasmid pMutin4 for the construction of the rocF BioBrick using 3 different melting temperatures in the Phusion PCR protocol, as well as to rerun the gel electrophoresis of the RocF fragments and the double terminator fragments obtained this morning.

Materials and Protocol

Please refer to PCR for the Phusion PCR protocol. The details for the 3 PCR reactions are mentioned below:

PCR

Tube Part to be amplified DNA fragment consisting the part Forward primer Reverse Primer Melting Temperature (Tm in °C) Size of the fragment (in bp) Extension time* (in seconds)
1 Pspacoid Promoter pMutin4 P1P1 forward P2P1 reverse 51 106 approx. 15
2 Pspacoid Promoter pMutin4 P1P1 forward P2P1 reverse 50 106 approx. 15
3 Pspacoid Promoter pMutin4 P1P1 forward P2P1 reverse 52 106 approx. 15

Table 2: Table represents 3 different Phusion PCR reactions for the amplification of Pspac_oid promoter, so that it can be ligated together with other fragments for the construction of rocF with the help of Gibson Cloning method.

  • The extension rate of the Phusion polymerase is 1Kb/ 30 seconds. Therefore the extension time of each PCR reaction is different.
  • To learn more about the rocF fragments, please refer to the Cloning strategy for rocF.

Result

Newcastle 050810 PCR 100bp second gel.png

Figure 2: Gel electrophoresis of the pSB1C3, Pspac_oid promoter, rocF fragments and double terminator.

  • Lane 1: 100bp DNA ladder
  • Lane 2: 1st fragment of rocF CDS
  • Lane 3: 2nd fragment of rocF CDS
  • Lane 4: 3rd fragment of rocF CDS
  • Lane 5: Double Terminator
  • Lane 6: Pspac_oid promoter (Tm 50°C i.e. Tube 2 of PCR reaction which is mentioned above)
  • Lane 7: Pspac_oid promoter (Tm 51°C i.e. Tube 1 of PCR reaction which is mentioned above)
  • Lane 8: Pspac_oid promoter (Tm 52°C i.e. Tube 3 of PCR reaction which is mentioned above)
  • Lane 9: 100bp DNA ladder


Pspac_oid pormoter 1st fragment of rocF CDS 2nd fragment of rocF CDS) 3rd fragment of rocF CDS Double Terminator
Size of the Fragment (in bp) 148 approx. 246 approx. 597 approx. 125 approx. 116 approx.

Table 3: Table represents the size of the fragments represented as bands on the gel in their corresponding lanes.

Discussion

Correct bands size was observed in all lanes. The three different melting temperature used during the PCR for the Pspac_oid promoter were successful.

Conclusion

The amplified fragments of RocF, double terminator and the Pspac_oid promoter have been successful.

Concrete Tensile Splitting Test

Aim

To obtain samples of cracked concrete for BacillaFilla to fill up the cracks and also to determine the tensile strength of concrete before the cracks are filled up.

Materials

  • Concrete cylinder
  • Jubilee clips

Procedure

  1. A concrete cylinder was made beforehand and left for more than 28 days to cure so that a straight line of crack will form down the diameter of the cylinder.
  2. The cylinder is placed on two diametrically opposed loading generators. Two pieces of plywood are placed between the loading plates and the concrete cylinder to prevent failure in compression.
  3. The generator is then started for loading until the cylinder forms a crack down the diameter. The maximum load is recorded and tensile strength of the concrete cylinder is calculated.
Concrete cylinder
Loading generator
Cracked concrete
Steven giving his baby to Phil
Failure load

Result

The maximum load that is recorded from the test is 171.3 kN.

Discussion

From the formula f=(2P)/(πBD), where f=tensile strength, P=Maximum applied load, B=Depth of cylinder, D=Diameter of cylinder,

With the depth and diameter of the cylinder as 30m and 15m respectively, we calculated the maximum tensile strength of this concrete cylinder to be 242.3 kN/m².

Conclusion

We now have the original tensile strength of the concrete cylinder, which is 242.3 kN/m². We will test its tensile strength again after the concrete has been filled up by BacillaFilla.
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