Category Archives: Abalone Withering Syndrome qPCR Troubleshooting

Abalone Withering Syndrome qPCR Troubleshooting

qPCR – Promega 2x GoTaq Probe Master Mix Withering Syndrome Assay Limit of Detection

Ran limit of detection qPCR for the Promega 2x GoTaq Probe Master Mix using p18RK7 (from 20120730) primary curve and p18RK7 low curve (from 20140507).

Master mix calcs are here: 20140506 – qPCR WSN1 Promega LoD-1

Cycling params, plate layout, etc. can be found in the qPCR Report in the Results below.

Results:

qPCR Report (PDF): Sam_2014-05-21 14-04-07_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-05-21 14-04-07_CC009827.pcrd

Data can be found here: Promega Master Mix Withering Syndrome Limit of Detection

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qPCR – p18RK7 Curve IDT Primer Test

Quick withering syndrome qPCR assay test using the ABI RLP_p probe and IDT primers. This is to see if we can still use IDT primers that have NOT been ABI HPLC purified. This would be a significant cost savings, as HPLC purified primers from ABI cost ~$60 each (“regular” IDT primers only cost ~$6 each). This should work.

Master mix calcs are here: 20140502 – qPCR p18RK7 Curve WSN1 IDT Primers

Used p18RK7 curve from 20120730 because we are running low on the p16RK7 curve (from the same date).

Plate layout, cycling params, etc can be found in the qPCR Report in the Results below.

Results:

qPCR Report (PDF): Sam_2014-05-02 09-42-38_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-05-02 09-42-38_CC009827.pcrd

Not surprising,the IDT primers work just fine. Will continue to use/order IDT primers for withering syndrome PCRs.

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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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Bacteria Cultures – p16RK3-A, B, & C Clones from 5/2004

We identified a usable clone for the RLP qPCR assay that contains the entire GenBank sequence (AF133090), instead of the partial (and incorrect) clone that Lisa and I had used previously (see 20120323); leading to a month’s worth of qPCRs that failed to produce the expected, and desired, results. This clone is vector pCR2.1 (Invitrogen) containing the full GenBank sequence, AF133090).

Since the p16RK3-C clone failed to grow yesterday, decided to make cultures from all three frozen stocks of p16RK3 to ensure that at least one will grow.

Three 5mL O/N cultures in LB+Amp (100ug/mL) were inoculated directly from the frozen stocks and grown at 37C on a rocker in a 15mL conical tube.

Results:

All three cultures failed to grow. Left cultures in incubator on rocker. Will make some LB and LB+Amp plates and try streaking out all three frozen stocks. Also will try growing the frozen stocks in liquid LB (no Amp) O/N in hopes of getting a culture for mini prep.

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