Team:Newcastle/5 August 2010

From 2010.igem.org

(Difference between revisions)
 
(118 intermediate revisions not shown)
Line 4: Line 4:
==Aim==
==Aim==
-
The aim of this experiment is to extract plasmid DNA pSB1C3, pSB1AK3 and plasmid containing ''lacI'' Biobrick from ''E. coli'' DH5α cells with the help of Qiagen miniprep kit and confirming the extraction with the help of nanodrop experiment.
+
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==
-
Please refer to: [[Team:Newcastle/Minipreps| Minipreps]] for Qiagen miniprep protocol, [[TeamNewcastleNanoDrop Spectrophotometer| Nanodrop Spectrophotometer]] for nanodrop protocol and [[TeamNewcastleRestriction digests| Restriction digests]] for restriction digestion protocol.
+
Please refer to: [[Team:Newcastle/Gel electrophoresis| Gel electrophoresis]].
==Result==
==Result==
-
* '''Lane 1''': 1kb DNA ladder
+
[[Image:Newcastle_050810_first_PCR_gel.png|400px]]
-
* '''Lane 2''': Extraction of pSB1C3 plasmid (No. 1)
+
 
-
* '''Lane 3''': Extraction of pSB1C3 plasmid (No. 2)
+
'''Figure 1''': Gel electrophoresis of the pSB1C3, Pspac_oid promoter, ''rocF'' fragments and double terminator.
-
* '''Lane 4''': Extraction of pSB1C3 plasmid (No. 3)
+
 
-
* '''Lane 5''': Extraction of pSB1C3 plasmid (No. 4)
+
* '''Lane 1''': 1 Kb DNA ladder
-
* '''Lane 6''': Extraction of plasmid containing lacI (No. 1)
+
* '''Lane 2''': BioBrick compatible vector pSB1C3
-
* '''Lane 7''': Extraction of plasmid containing lacI (No. 2)
+
* '''Lane 3''': Pspac_oid promoter
-
* '''Lane 8''': Extraction of pSB1AK3 plasmid containing double terminator (No. 1)
+
* '''Lane 4''': 1st fragment of ''rocF'' CDS
-
* '''Lane 9''': Extraction of pSB1AK3 plasmid containing double terminator (No. 2)
+
* '''Lane 5''': 2nd fragment of ''rocF'' CDS
 +
* '''Lane 6''': 3rd fragment of ''rocF'' CDS
 +
* '''Lane 7''': Double Terminator
 +
* '''Lane 8''': 1 Kb DNA ladder
{|border=1
{|border=1
|-
|-
-
!'''pSB1C3  
+
!
-
(No. 1)'''
+
!'''Biobrick compatible vector pSB1C3'''
-
!'''pSB1C3
+
!'''Pspac_oid pormoter'''
-
(No. 2)'''
+
!'''1st fragment of ''rocF'' CDS'''
-
!'''pSB1C3
+
!'''2nd fragment of ''rocF'' CDS)'''
-
(No. 3)'''
+
!'''3rd fragment of ''rocF'' CDS'''
-
!'''pSB1C3
+
!'''Double Terminator'''
-
(No. 4)'''
+
-
!'''lacI
+
-
(No. 1)'''
+
-
!'''lacI
+
-
(No. 2)'''
+
-
!'''Double terminator
+
-
(No. 1)'''
+
-
!'''Double terminator
+
-
(No. 2)'''
+
|-
|-
-
|44.0 µl/ml
+
|'''Size of the Fragment (in bp)'''
-
|19.9 µl/ml
+
|2072 approx.
-
|25.0 µl/ml
+
|148 approx.
-
|30.8 µl/ml
+
|246 approx.
-
|10.0 µl/ml
+
|597 approx.
-
|44.2 µl/ml
+
|125 approx.
-
|9.2 µl/ml
+
|116 approx.
-
|39.7 µl/ml
+
|}
|}
-
'''Table 1''': Nanodrop spectrophotometer experiment result. Table represents the amount of plasmid present in µl/ml quantity.
+
'''Table 1''': Table represents the size of the fragments represented as bands on the gel in their corresponding lanes.
==Discussion==
==Discussion==
-
We found bands in the lane 2, 3, 4, 5 and 6 showing the presence of plasmid in ''E. coli'' DH5α cells. The ideal concentration of DNA calculated using nanodrop experiment is 150 µg/ml but in the table 1, where all the values have been less than 150 µg/ml which shows that even though there is plasmid present in the cells but it is present in very low amount. Also while doing nanodrop experiment, we measured 260/280 nm ratio for all the samples came out to be between 2.0 to 2.4 approximately which shows that there is a high RNA contamination in the samples.
+
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 there is plasmid present in the ''E. coli'' DH5α cells but they are present in a very low amount and having high RNA contamination possibly due to the following reasons:
+
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:
-
# P1 buffer which contains RNAse might be contaminated.
+
# Primer sequences could be incorrect.
-
# RNAse enzyme might have gotten inactive.
+
# Melting temperature could be incorrect.
 +
# Plasmid pMutin4 could have degenerated due to long term storage.
==Solution for the problem==
==Solution for the problem==
-
# If P1 buffer of the Qiagen miniprep kit is contaminated, then use a different kit. We have Promega miniprep kit which will be used tomorrow.
+
# Check the primer sequences so as to eliminate any problems associated with the primer sequence.
-
# If RNAse enzyme is inactive, then add extra RNAse into the P1 buffer. We would be adding 10 µl in the P1 buffer solution.
+
# Perform PCR reactions for the Pspac_oid fragment with 3 different melting temperatures at 50°C, 51°C and 52°C.
-
{{Team:Newcastle/footer}}
+
=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 [[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==
 +
Please refer to  [[Team:Newcastle/PCR| PCR]] for the Phusion PCR protocol. The details for the 3 PCR reactions are mentioned below:
 +
 
 +
===PCR===
 +
{|border=1
 +
|-
 +
!'''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 [[Media:Cloning_strategy_for_rocF.pdf|Cloning strategy for ''rocF'']].
 +
 
 +
==Result==
 +
[[Image:Newcastle_050810_PCR_100bp_second_gel.png|500px]]
 +
 
 +
'''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
 +
 
 +
 
 +
{|border=1
 +
|-
 +
!
 +
!'''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==
 +
 
 +
# 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_Concrete_8.jpg|thumb|Cracked concrete]]
 +
|[[Image:Newcastle_Concrete_9.jpg|thumb|Steven giving his baby to Phil]]
 +
|}
 +
 
 +
[[Image:Newcastle Concrete 10.jpg|thumb|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.

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.
Retrieved from "http://2010.igem.org/Team:Newcastle/5_August_2010"