Contents 1 Gel Electrophoresis for the amplified fragments of Pspac_oid promoter and lacI 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 Result 2.4 Discussion 2.5 Conclusion 3 Gel Electrophoresis for Amplified Pspac_oid promoter 3.1 Aim 3.2 Materials and Protocol 3.3 Result 3.4 Discussion 3.5 Conclusion 4 lacI ligation (repeat) 4.1 Aim 4.2 Materials and Protocols 4.3 Discussion 4.4 Results 5 lacI and pVeg spoVG Transformation 5.1 Aim 5.2 Materials and Protocol 5.3 Discussion 5.4 Conclusion

Gel Electrophoresis for the amplified fragments of Pspac_oid promoter and lacI

Aim

The aim of the experiment is to perform gel electrophoresis for the two PCR reactions, lacI and Pspac_oid promoter that were done using a different stock of plasmid pMutin4 on 5th August, 2010.

Materials and Protocol

Please refer to: Gel electrophoresis.

Result

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

• Lane 1: 1kb DNA ladder
• Lane 2: Plamid pMutin4 containing lacI
• Lane 3: Plamid pMutin4 containing Pspac_oid promoter
• Lane 4: 100bp DNA ladder

	Pspac_oid promoter	lacI
Size of the Fragment (in bp)	106 approx.	1400 approx.

Table 1: Table represents the size of the fragments represented as bands on the gel in their corresponding lanes.

Discussion

A correct sized band was observed in lane 2, lacI which serve as the positive control. However no band was observed in lane 3, which contain the Pspac_oid promoter.

Conclusion

This experiment shows that the plasmid pMutin4 is intact as the lacI frangment have been successfully amplified (Lane 2). However the Pspac_oid promoter are still not being amplified. This could be due to the following reason:

1. Melting temperature could be incorrect.

Solution for the problem

1. Try a range of melting temperature.

Amplification of Pspac_oid promoter by PCR

Aim

The aim of this experiment is to amplify the Pspac_oid promoter 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 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	pMutin4	P1P1 forward	P2P1 reverse	49	106 approx.	15
2	Pspacoid Promoter	pMutin4	P1P1 forward	P2P1 reverse	50	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.

Result

Figure 2: Gel electrophoresis of the Pspac_oid promoter from plasmid pMutin4.

• Lane 1: 100bp DNA ladder
• Lane 2: Plamid pMutin4 containing Pspac_oid promoter at 49°C
• Lane 3: Plamid pMutin4 containing Pspac_oid promoter at 50°C
• Lane 4: 100bp DNA ladder

	Pspac_oid promoter at 49°C	Pspac_oid promoter at 50°C
Size of the Fragment (in bp)	106 approx.	106 approx.

Table 3: Table represents the size of the fragments represented as bands on the gel in their corresponding lanes.

Discussion

We found two very faint bands in the lanes 2 and 3.

Conclusion

Today afternoon, we would be running gel electrophoresis to check the outcome of the 2 PCR reactions and later all the fragments will be ligated with help of Gibson protocol if the fragments have amplified.

Gel Electrophoresis for Amplified Pspac_oid promoter

Aim

The aim of the experiment is to perform gel electrophoresis for the two PCR reactions for Pspac_oid promoter which took place today morning and thus confirm that they were successful.

Materials and Protocol

Please refer to: Gel electrophoresis.

Result

Figure 2: Gel electrophoresis of the Pspac_oid promoter from plasmid pMutin4.

• Lane 1: 100bp DNA ladder
• Lane 2: Plamid pMutin4 containing Pspac_oid promoter at 49°C
• Lane 3: Plamid pMutin4 containing Pspac_oid promoter at 50°C
• Lane 4: 100bp DNA ladder

	Pspac_oid promoter at 49°C	Pspac_oid promoter at 50°C
Size of the Fragment (in bp)	106 approx.	106 approx.

Table 3: Table represents the size of the fragments represented as bands on the gel in their corresponding lanes.

Discussion

We found two very faint bands in the lanes 2 and 3.

Conclusion

This experiment shows that the PCR reaction was unsuccessful even though we got a faint band for Pspac_oid promoter. This time, we are going to use plasmid containing KinA Biobrick and plasmid containing stochastic switch Biobrick which were devised by Team Newcastle 2009. We would be dealing with this on Monday 9th August, 2010.

lacI ligation (repeat)

Aim

We aim to repeat the ligation from week 12-17th July.

Materials and Protocols

Please refer to ligation for protocol. However, we did not do a positive control for this experiment.

Discussion

On Monday we will transform E.coli with the ligation product.

Results

Please refer to 09.08.10 for results.

lacI and pVeg spoVG Transformation

Aim

We aim to transform competent E.coli DH5α with pVeg spoVG and lacI in preparation for Gibson Cloning.

Materials and Protocol

Please refer to transformation for protocol.

Discussion

We expect cells transformed with lacI and pVeg to survive on the ampicillin plates.

Conclusion

Please refer to 09.08.10 for results.