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Contents 1 Gel Electrophoresis for the Amplified Fragments of rocF 1.1 Aim 1.2 Materials and Protocol 1.3 Result 1.4 Discussion 1.5 Conclusion 1.6 Solution for the problem 2 Amplification of the Pspac_oid promoter and RocF fragments by PCR 2.1 Aim 2.2 Materials and Protocol 2.2.1 PCR 2.3 Result 2.4 Discussion 2.5 Conclusion 3 Concrete Tensile Splitting Test 3.1 Aim 3.2 Materials 3.3 Procedure 3.4 Result 3.5 Discussion 3.6 Conclusion 4 Gel Electrophoresis for the Amplified Fragments of rocF 4.1 Aim 4.2 Materials and Protocol 4.3 Result 4.4 Discussion 4.5 Conclusion 4.6 Solution for the problem 5 Amplification of Pspac_oid promoter and lacI from a different stock of pMutin4 by PCR 5.1 Aim 5.2 Materials and Protocol 5.2.1 PCR 5.3 Discussion 5.4 Conclusion

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

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

• Lane 1: 1kb 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: 1kb 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.	106 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

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)	106 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 lanes 2,3,4 and 5 but not in lanes 6,7, and 8. The three different melting temperature used during the PCR for the Pspac_oid promoter is not successful.

Conclusion

The amplified frangments of RocF and the double terminator have been successful. However we are still not able to obtain the Pspac_oid promoter even when we have checked the primer sequence and using 3 different melting temperature. This could be due to the folloeing reason:

1. Plasmid pMutin4 could have degenerated due to long term storage.

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

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

Result

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

Discussion

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: Gel electrophoresis.

Result

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)	106 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

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.

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:

1. Plasmid pMutin4 could have degenerated due to long term storage.

Solution for the problem

1. Use a different stock of the plasmid pMutin4 and perform PCR reaction for the amplification of Pspac_oid promoter.

Amplification of Pspac_oid promoter and lacI from a different stock of pMutin4 by PCR

Aim

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

Please refer to PCR for 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	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.

• The extension rate of the Phusion polymerase is 1Kb/ 30 seconds. Thus the extension time of each and every PCR reaction is slightly different.

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.

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.