PCR – Full Length AFF133090 (Abalone Withering Syndrome 16s)

Ran conventional PCR to amplify the full length abalone withering syndrome 16s product for subsequent cloning. Used four different DNA plasmid preps as template, for comparison purposes. Plasmid preps used were:

  • p16RK7 (from 20120718)
  • p18RK7 (from 20120718)
  • pWC8 (from 20120718)
  • p16RK7 A (from 20131106)

Primers used:

  • WS_16s_1_F
  • WS_16s_1501_R

Master mix calcs are here: 20131203 – cPCR WS Full Length

All reactions were run in duplicate.

Cycling params:

  • 95C – 10m

40 cycles of:

  • 95C – 15s
  • 55C – 15s
  • 72C – 2m

Results:

Ran 20uL (of 50uL reaction) of each sample on 0.8% TBE gel.

Lanes (left ro right):

1 – Hyperladder I (Bioline)

2 – p16RK7 (from 20120718)

3 – p16RK7 (from 20120718)

4 – p18RK7 (from 20120718)

5 – p18RK7 (from 20120718)

6 – pWC8 (from 20120718)

7 – pWC8 (from 20120718)

8 – p16RK7 A (from 20131106)

9 – p16RK7 A (from 20131106)

10 – NTC

11 – NTC

Expected a band of ~1500bp. Bands of all samples (except pWC8) run at ~1500bp. pWC8 runs a bit higher and will not be used for cloning, as part of our troubleshooting the failure of our plasmid qPCR standard curve for withering syndrome.

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