Contents 1 Subtilin Immunity 1.1 Gel Electrophoresis and Gel Extraction 1.1.1 Aims 1.1.2 Materials and protocol 1.1.3 Results 1.1.4 Discussion 1.1.5 Conclusion 1.2 PCR of pSB1C3 for rocF and Subtilin Immunity 1.2.1 Aims 2 Gel Extraction 2.1 Aims 2.2 Materials and Protocol 2.3 Results 2.4 Discussion 2.5 Conclusion 3 Miniprep of yneA, pGFPrrnB and pSB1C3 3.1 Aim 3.2 Materials and Protocol 3.3 Results 3.4 Discussion 3.5 Conclusion 4 Transformation of Ligated Products 4.1 Aims 4.2 Materials and Protocol 5 Ligation of yneA with Vectors 5.1 Aims 5.2 Materials and Protocol 5.3 Results, Discussion and Conclusion

Subtilin Immunity

Gel Electrophoresis and Gel Extraction

Aims

Following yesterday's Phusion PCR reactions, we plan to first perform gel electrophoresis of the amplified DNA sequences to check if they are of the correct sizes. If they appear to be successful we will then perform gel extraction, and finally nanodrop to record the level of DNA.

Materials and protocol

Please refer to the:

• gel electrophoresis,
• gel extraction and
• NanoDrop spectrophotometer protocols.

Results

Figure #. Gel electrophoresis of the amplified PCR products

Lane 1: 1 Kb ladder Lane 2: pSB1C3 (for Subtilin Immunity) Lane 3: pSB1C3 (for rocF)

Lane 4: pVeg (for Subtilin Immunity) Lane 5: terminator (for Subtilin Immunity) Lane 6: pSpacoid (for rocF) Lane 7: terminator (for rocF) Lane 8: 100 bp ladder

Discussion

When we saw the results from yesterday's Phusion PCR, the pSBIC3 vector did not show any band. Therefore, we decided to make a range of Tms to perform another set of PCR reactions. Three Tms were used: 60°C, 65°C and 70°C. Six tubes were set up; a duplicate was created for each Tm. After the PCR reactions were finished, there still wasn't any visible pSBIC3 vector band showing up. The problem could have occurred due to the working stocking solution for our primers. Our next step was to re-make the working stock solution for our primers. Also, we had decided to use the Tms 55°C, 60°C, 65°C, 70°C - duplicate for each, making eight PCR tubes in total. The PCR machines were left to run and results shall be obtained on Monday.

Conclusion

PCR of pSB1C3 for rocF and Subtilin Immunity

Aims

Gel Extraction

Aims

To purify the PCR products of digested yneA, pGFPrrnB and pSB1C3 from yesterday.

Materials and Protocol

Please refer to gel extraction and nanodrop.

Results

Discussion

Conclusion

Miniprep of yneA, pGFPrrnB and pSB1C3

Aim

To produce stocks of yneA, pGFPrrnB and pSB1C3.

Materials and Protocol

Please refer to miniprep and nanodrop.

Results

Discussion

Conclusion

Transformation of Ligated Products

Aims

To produce more colonies of the ligated products of yneA with pGFPrrnB and pSB1C3.

Materials and Protocol

Please refer to transformation of E. coli.

Ligation of yneA with Vectors

Aims

To ligate yneA with pGFPrrnB and yneA with pSB1C3 (A repeat of yesterday.

Materials and Protocol

Please refer to ligation.

Results, Discussion and Conclusion

Please refer to 23.08.10.