Tag Archives: Martha’s Vineyard

qPCRs – MV hemocyte cDNA from 20090614

qPCR – CIAPIN Primers

Set up qPCR with Cv_CIAPIN_F/R primers. Plate layout/PCR set up is here.

Results: Waters are clean. gDNA samples did not amplify. To be analyzed later with other genes that Mac has run.

 

qPCR – CatY Primers

Set up qPCR with Cv_CatY_F/R primers. Plate layout/PCR set up is here.

Results: Waters are clean. gDNA samples did not amplify. To be analyzed later with other genes that Mac has run.

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qPCR – MV hemocyte cDNA: Test Immomix (SYTO13) vs. Strategene SYBR

Due to craziness seen in melting curves, fluorescence, and empty wells from the previous run, will compare SYTO vs. SYBR with select MV cDNAs. Additionally, acquired some qPCR strip caps to use instead of the ABI film. Used Cv_18s_F/R primers. qPCR set up/plate layout is here.

Results: Both seem to work fine. H2O fluorescence is weird, but doesn’t come up in the melting curves. Strategene SYBR provides a brighter signal, but results in a higher melting temp than the SYTO.

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qPCR – MV hemocyte cDNA from yesterday

qPCR set up/plate layout is here. Used Cv_18s_F/R primers to assess samples’ “useability” for future qPCRs. Used an ABI optically clear adhesive film instead of caps. Ran out of appropriate caps.

Results: Yep, seal was bad. Explains most of the weirdness seen. However, will compare SYTO and Strategene SYBR.

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qPCR – DNased MV hemocyte RNA from earlier today AND Turbo kit test

qPCR set up/plate layout is here. Used Cv_18s_F/R primers for the MV hemocyte RNA and Gigas_18s_F/R primers for the Turbo kit test. Anneal 55C.

Results: DNase treatment worked on all but the following samples: B23, B14, A21. However, these three samples were slightly below the initial, background fluorescence in each sample. The Turbo kit test indicates that all three kits are working perfectly and all can/should be used with confidence for treating samples.

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DNase Treatment – MV hemocyte RNA from yesterday

Samples 3326: B23, A25, A22, B14, A21, A10 B22 came up positive for gDNA still. These were retreated according to Ambion protocol with a brand new Turbo DNA-free DNase kit. Additionally, I tested all three existing kits by “spiking” 19uL of H2O with 1uL (~200ng) of gigas gDNA; one tube for each kit and an untreated sample. Will qPCR to see if gDNA removal was successful.

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DNase Treatment – MV hemocyte RNA from earlier today

The entire 20uL of RNA were treated with Ambion’s Turbo DNA-free kit according to protocol and spec’d.

Mac performed qPCR on the DNase-treated RNAs to verify removal of residual gDNA.

Results: Samples 3326: B23, A25, A22, B14, A21, A10 B22 came up positive for residual gDNA. Will retreat these samples and qPCR again.

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