Restriction Digest

Performed restriction digest with NcoI (NEB) to linearize plasmids prepared earlier today to use in the RLP (withering syndrome) qPCR assay. Reactions were set up as follows, incubated @ 37C for two hours and then heat-inactivated @ 65C for 10mins. 2uL of undigested DNA and digested DNA were run on a 1% modified TAE gel.

p16RK3-A

DNA (1.36ug) – 10uL

10x Buffer 3 – 2.8uL

H2O – 14.7uL

NcoI – 0.5uL

p16RK3-C

DNA (873.6ng) – 24uL

10x Buffer 3 – 2.8uL

H2O – 0.7uL

NcoI – 0.5uL

Results:

Gel Layout (from left to right):

Lane 1 – Hyperladder I (Bioline)

Lane 2 – Undigested p16RK3-A

Lane 3 – NcoI digested p16RK3-A

Lane 4 – Undigested p16RK3-C

Lane 5 – NcoI digested p16RK3-C

Surprisingly, the NcoI digests resulted in TWO bands instead of the expected SINGLE band. The bands are ~2500bp and ~3000bp, which totals ~5500bp for the construct. However, I was not expecting two bands, as I was told to use NcoI since it would linearize the plasmid. Clearly, this is not the case.

Looking at the AF133090 sequence and the pCR2.1 TOPO sequence, it appears that there is a NcoI site in each. As such, we end up with two bands, as seen in the gel above.

If the AF133090 sequence is cloned in the direction that it exists in GenBank (with the NcoI site at base 1366), then we should see fragments of 1720bp (which contains most of the AF133090 sequence) and 3712bp (which is mostly vector). However, that’s not what we see in the gel, suggesting that the AF133090 sequence is cloned in reverse.

If that is the case, then we would expect fragments of 3052bp (which contains most of the AF133090 sequence) and 2379bp (which is mostly vector). This is exactly what we see on this gel.

So, the higher molecular weight band contains our sequence of interest.

After speaking with Carolyn, she has suggested that I run half of this reaction on a gel and purify the higher molecular weight band. We will then compare the use of this NcoI digest and the gel-purified fragment in the standard curves for the RLP qPCR assay to see if there’s any difference.

Mini Prep – Bacterial Cultures from 20120409 & 20120410

Isolated plasmid DNA from cultures inoculated on 20120409 (p16RK3-C) & 20120410 (p16RK3-A). These cultures finally produced bacteria. Slow growth was likely due to anaerobic conditions (sealed 15mL conical tubes) and slow agitation (on a rocker instead of a shaker). Isolated plasmid DNA using Qiagen Mini Prep Spin Kit according to manufacturer’s protocol. Eluted plasmid DNA with 30uL of Buffer EB and quantified on Roberts Lab NanoDrop1000.

Results:

p16RK3-A = 134.2ng/uL

p16RK3-C = 36.4ng/uL

Bacteria Culture – p16RK3-C RLP Clone 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).

A 5mL O/N culture in LB+Amp (100ug/mL) was inoculated directly from the frozen stock and grown at 37C on a rocker in a 15mL conical tube.

Results:

Culture failed to grow. Will try again, but will inoculate multiple cultures from multiple stocks to ensure that at least one will grow up. Left culture on rocker in incubator.