pSB1C3 plasmid gel electrophoresis

Aims

The aim of this experiment is to run gel electrophoresis for the extracted and linearized plasmid pSB1C3 fragment which were amplified at 4 different melting temperatures by 4 separate PCR reactions.

Materials and Protocol

Please refer to: gel electrophoresis for gel electrophoresis protocol.

Result

Figure 1: Gel electrophoresis of the amplified linearized plasmid pSB1C3 fragments ran at 4 different melting temperatures, Tms, (50, 60, 65, 70°C). A 1 kb DNA ladder was used on either side of lanes.

• Lane 1: pSB1C3 fragment amplified at 55°C
• Lane 2: pSB1C3 fragment amplified at 60°C
• Lane 3: pSB1C3 fragment amplified at 65°C
• Lane 4: pSB1C3 fragment amplified at 70°C
• Lane 5: pSB1C3 fragment amplified at 55°C
• Lane 6: pSB1C3 fragment amplified at 60°C
• Lane 7: pSB1C3 fragment amplified at 65°C
• Lane 8: pSB1C3 fragment amplified at 70°C

Discussion

No bands were found in any of the lanes. Yesterday, a faint band was found when the melting temperature was set at 65°C but today no band is found in lane 3 and lane 7. This makes finding the cause for no amplification even difficult. We would still be looking into it and would be changing other parameters.

Conclusion

The PCR reaction failed as there is no amplification found in any of the reactions.

pSB1C3 plasmid gel electrophoresis by adding EtBr

Aims

The aim of this experiment is to run gel electrophoresis for the extracted and linearized plasmid pSB1C3 fragment which were amplified at 4 different melting temperatures by 4 separate PCR reactions and to put Ethidium Bromoide (EtBr) in the agarose gel instead of safeview die so as to get better resolution and brighter bands.

Materials and Protocol

Please refer to: gel electrophoresis for gel electrophoresis protocol.

Please note that in this protocol we have made a minor modification which is that instead of adding 5µl of safeview dye, we have added 5µl of EtBr into the agarose gel so as to get better resolution and brighter bands.

Result

Figure 2: Gel electrophoresis (containing EtBr) of the amplified linearized plasmid pSB1C3 fragments ran at 4 different melting temperatures, Tms, (50, 60, 65, 70°C). A 1 kb DNA ladder was used on either side of lanes.

• Lane 1: pSB1C3 fragment amplified at 55°C
• Lane 2: pSB1C3 fragment amplified at 60°C
• Lane 3: pSB1C3 fragment amplified at 65°C
• Lane 4: pSB1C3 fragment amplified at 70°C
• Lane 5: pSB1C3 fragment amplified at 55°C
• Lane 6: pSB1C3 fragment amplified at 60°C
• Lane 7: pSB1C3 fragment amplified at 65°C
• Lane 8: pSB1C3 fragment amplified at 70°C

Discussion

No bands were found in any of the lanes. Yesterday, a faint band was found when the melting temperature was set at 65°C but today no band is found in lane 3 and lane 7. This makes finding the cause for no amplification even difficult. We would still be looking into it and would be changing other parameters.

Conclusion

The PCR reaction failed as there is no amplification found in any of the reactions.

Amplification of Pspac_oid promoter by PCR

Aim

The aim of this experiment is to amplify 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 with the help of 4 different 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 PromoterPlease 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. 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 4 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 4 PCR reactions and later all the fragments will be ligated with help of Gibson protocol if the fragments have amplified.