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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 Pspac_oid promoter by PCR 2.1 Aim 2.2 Materials and Protocol 2.2.1 PCR 2.3 Discussion 2.4 Conclusion 3 Concrete Tensile Splitting Test 3.1 Aim 3.2 Materials 3.3 Procedure 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

Gel Electrophoresis for the Amplified Fragments of rocF

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.

Materials and Protocol

Please refer to: Gel electrophoresis.

Result

• 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

We found bands in the lanes 2,4,5,6 and 7 of the correct sizes but lane 3 did not contain any band.

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:

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. 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.
2. If RNAse enzyme is inactive, then add extra RNAse into the P1 buffer. We would be adding 10 µl in the P1 buffer solution.

Amplification of Pspac_oid promoter by PCR

Aim

The aim of today's experiment is to amplify Pspac_oid promoter fragment from plasmid pMutin4 for the construction of rocF BioBrick with the help of 3 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	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. Thus the extension time of each and every PCR reaction is slightly different.
• For learning about the rocF fragments, please refer to the Cloning strategy for rocF.

Discussion

All the 3 Phusion PCR reactions were done however, gel electrophoresis was 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 and after the cracks are filled up.

Materials

• Concrete cylinder
• Jubilee clips

Procedure

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

• 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

	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

We found bands in the lanes 2,4,5,6 and 7 of the correct sizes but lane 3 did not contain any band.

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:

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. 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.
2. If RNAse enzyme is inactive, then add extra RNAse into the P1 buffer. We would be adding 10 µl in the P1 buffer solution.