On Friday, October 27th and 28th, the team worked on extracting DNA from agarose gels coming from PCR amplified field samples. The same samples that had tested positive in the Summer banded at the mip and 16s molecular weights once more. Please refer to the pictures and tables for more info.
Agarose gel DNA Extraction for sequencing (10-17-15)
Utilized UV lamp to visualize bands. Cut with scalpel and placed in DNAase free
tubes.
Extracted DNA from gel as per the Thermo Scientific GeneJET Extraction kit:
Added binding buffer on 1mg (agarose):1μL ratio.
Incubated at 55◦C for ≈12 min (until get dissolved). Inverting at 2 min intervals.
Vortexed for 5-10 seconds.
For mip DNA only, a 1 gel volume of 100% isopropanol was added.
Transferred solution to purification column and centrifuged for 1 min at 13.2 rpm.
Flow through was discarded.
Added 100µL of Binding Buffer to column and centrifuged for 1 min at 13.2 rpm.
Discarded flow through. And centrifuged column an additional minute.
Transferred column to DNA free centrifuge tube and added 50µL of Elution Buffer.
Centrifuged for 1 minute at 13.2 rpm. Column was discarded and DNA stored at -
20°C.
mip gel
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Lane 1
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Lane 2
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Lane 3
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Lane 4
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Lane 5
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Lane 6
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Lane 7
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Lane 8
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MW
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mip +
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empty
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12w
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13w
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14w
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15w
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16w
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mip and 16s gel
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Lane 1
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Lane 2
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Lane 3
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Lane 4
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Lane 5
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Lane 6
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Lane 7
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Lane 8
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MW
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m+
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empty
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9b mip
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17w mip
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9b 16s
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17w 16s
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16s +
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16s gel
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Lane 1
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Lane 2
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Lane 3
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Lane 4
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Lane 5
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Lane 6
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Lane 7
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Lane 8
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MW
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16s +
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empty
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12w
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13w
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14w
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15w
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16w
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