Following [[Team:Newcastle/19_August_2010| 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.
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===Materials and protocol===
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Please refer to the:
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*[[Team:Newcastle/Gel_electrophoresis| gel electrophoresis]], [[Image:Newcastle Gel 1 (20-08-10).jpg|right|300px|The gel loaded up with the amplified PCR products (blue) and the ladders on either side (green), carried out to check the size of the sequences. Please see results section below for key.]]
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*[[Team:Newcastle/Gel_extraction| gel extraction]] and
[[Image:Newcastle Gel 20-08-2010.jpg|700px|centre]]
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'''Figure 1''': Gel electrophoresis of the amplified PCR products
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*'''Lane 1''': 1 Kb ladder
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*'''Lane 2''': pSB1C3 (for Subtilin Immunity)
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*'''Lane 3''': pSB1C3 (for ''rocF'')
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*'''Lane 4''': pVeg (for Subtilin Immunity)
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*'''Lane 5''': terminator (for Subtilin Immunity)
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*'''Lane 6''': pSpacoid (for ''rocF'')
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*'''Lane 7''': terminator (for ''rocF'')
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*'''Lane 8''': 100 bp ladder
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===Discussion===
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After looking at the results of our gel, there are no distinct band in lanes 2-3, therefore pSB1C3 vector did not work for both Subtilin Immunity or ''rocF''. However, there are single bands in lanes 4-7 at similar positions to each other. They are about ... in size which is what is expected.
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[[Image:Newcastle Gel 2 (20-08-10) - extraction.jpg|right|300px]]
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[[Image:Newcastle Gel 3 (20-08-10) - extraction.jpg|300px|right]]
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As lanes 2-3 showed no bands, we decided to make a range of Tms to perform another set of PCR reactions to check whether the Tm was the problem. Three Tms were used: 60°C, 65°C and 70°C and 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.
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As lanes 4-7 had bands at the right positions, gel extraction of these four parts was carried out. The concentration of DNA was then measured using the Nanodrop spectrophotometer.
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===Conclusion===
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The PCR machines were left to run and results of the eight PCR reactions at the 4 different temperatures shall be obtained on [[Team:Newcastle/23_August_2010| Monday]]. After a test gel has been run and if bands appear at the right sizes then gel extraction will be carried out.
Has pMutin4 (and thus lacI) integrated into the chromosome...will be useful for characterisation.. will be transforming this strain with our filamentous cell and arginase BioBricks.
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