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Contents 1 Amplification of Pspac_oid promoter by PCR 1.1 Aim 1.2 Materials and Protocol 1.2.1 PCR 1.3 Discussion 2 Amplification of Pspac_oid promoter by PCR 2.1 Aim 2.2 Materials and Protocol 2.2.1 PCR 2.3 Discussion 3 Gel Electrophoresis for the amplified Pspac_oid promoter 3.1 Aim 3.2 Materials and Protocol 3.3 Result 3.4 Discussion 3.5 Conclusion

Amplification of Pspac_oid promoter by PCR

Aim

The aim of this experiment is to amplify the Pspac_oid promoter fragment from plasmid pMutin4 for the construction of rocF BioBrick using Phusion PCR.

Materials and Protocol

Please refer to PCR for Phusion PCR protocol. The details for the 4 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	58	106 approx.	15
2	Pspacoid Promoter	pMutin4	P1P1 forward	P2P1 reverse	59	106 approx.	15

Table 1: Table represents 4 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.

Discussion

For the gel electrophoresis results, please refer to the bottom section.

Amplification of Pspac_oid promoter by PCR

Aim

The aim of this experiment is to amplify the Pspac_oid promoter fragment from plasmid pMK-RQ containing Biobrick kinA and plasmid pMK-RQ containing stochastic switch developed by Team Newcastle 2009 for the construction of rocF BioBrick using Phusion PCR.

Materials and Protocol

Please refer to PCR for Phusion PCR protocol. The details for the 2 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	Plasmid containing kinA	P1P1 forward	P2P1 reverse	58	106 approx.	15
2	Pspacoid Promoter	Plasmid containing kinA	P1P1 forward	P2P1 reverse	59	106 approx.	15
3	Pspacoid Promoter	Plasmid containing stochastic switch	P1P1 forward	P2P1 reverse	58	106 approx.	15
1	Pspacoid Promoter	Plasmid containing stochastic switch	P1P1 forward	P2P1 reverse	59	106 approx.	15

Table 2: Table represents 2 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.

Discussion

For the gel electrophoresis results, please refer to the bottom section.

Gel Electrophoresis for the amplified Pspac_oid promoter

Aim

The aim of the experiment is to perform gel electrophoresis for the two PCR reactions Pspac_oid promoter amplified from plasmid pMutin4 and from plasmid pMK-RQ containing Biobrick kinA and plasmid pMK-RQ containing stochastic switch developed by Team Newcastle 2009.

Materials and Protocol

Please refer to: Gel electrophoresis.

Result

Figure 1: Gel electrophoresis of the lacI and Pspac_oid promoter.

• Lane 1: 100bp DNA ladder
• Lane 2: Plamid pMutin4 containing Pspac_oid promoter
• Lane 3: Plamid pMutin4 containing Pspac_oid promoter
• Lane 4: Plamid pMK-RQ (kinA BioBrick) containing Pspac_oid promoter
• Lane 5: Plamid pMK-RQ (kinA BioBrick) containing Pspac_oid promoter
• Lane 6: Plamid pMK-RO (stchastic switch BioBrick) containing Pspac_oid promoter
• Lane 7: Plamid pMK-RO (stchastic switch BioBrick) containing Pspac_oid promoter
• Lane 8: 100bp DNA ladder

	Pspac_oid pormoter
Size of the Fragment (in bp)	148 approx.

Table 3: Table represents the size of the Pspac_oid fragment represented as bands on the gel in all the lanes.

Discussion

Two bands were found in all the lanes which is of approximately 150 bp and 80 in size.

Conclusion

This experiment shows that the PCR reaction was not successful for the Pspac_oid fragment. The 80 bp band might be primer dimers. When the primer sequence was checked again, we found that there is a high complementarity within the primers itself and thus the amplification of the Pspac_oid fragment is not taking place due to the primer pairs joining together. The 150 bp band could be that of the Pspac_oid fragment. Therefore we will be doing gel extraction tomorrow to confirm if the 150 bp band is the Pspac_oid promoter.