PCR – Linearized pCR2.1/AF133090 Plasmid from 20120430

Ran PCRs using both the AF133090 full-length primers (WS_16s_1_F, WS_16s_1501_R) and the WSN primers (WSN1_F, WSN1_R). This time used the linearized pCR2.1/AF133090 plasmid as template instead of the bacterial colonies that were selected from cloning. Master mix calcs are here. Cycling params are as follows:

95C – 10m

40 cycles of:

  • 95C – 10s
  • 55C – 10s
  • 72C – 1.75m

Results:

Gel Loading:

Lane 1 – Hyperladder I (Bioline)

Lane 2 – 16s primers

Lane 3 – 16s primers NTC

Lane 4 – WSN primers

Lane 5 – WSN primers NTC

Essentially, this is the same result as the colony check from 20120503. The appropriate sized band (~1500bp) is generated using the 16s primers, but no band is generated using the WSN primers. Yes, there is contamination present in the 16s NTC sample, but this point is moot since the WSN primers still fail to generate a PCR product.

So, I have absolutely no idea what is cloned into this vector, despite the fact that primers designed on GenBank AF133090 produce a band.

I’m essentially running out of ideas on what else to do to get this working again. Will ask Lisa to make a curve from the linearized plasmid stock from DATE (not the pCR2.1/AF133090 plasmid that is being tested here) and see if she can get it to work. Also, Carolyn has suggested trying to run some of the more recent curves on a different qPCR machine. Will contact the guy in Health Sciences who has a Stratagene that we are allowed to use.

Plasmid Isolation & Restriction Digestion – pCR2.1/AF133090

Plasmid Isolation

Colony #2 from 20120426

Inoculated 5mL of 1x LB + Amp (100ug/mL) with colony #2 based off the of the PCR screening on 20120426 in a 50mL conical tube. Incubated @ 37C, 200RPM O/N. Plasmid DNA was isolated using Qiagen’s Mini Prep Spin Kit. Plasmid was eluted with 50uL of Buffer EB and spec’d on the Roberts Lab NanoDrop 1000.

Results:

[Plasmid] = 288.5ng/uL

Looks good; will proceed with linearization.

 

Restriction Digestion

Performed a restriction digestion using HindIII (NEB) @ 37C for 2hrs and then heat inactivated for 20mins @ 65C. Recipe:

  • Plasmid (1154ng): 4uL
  • 10x Buffer 2: 5uL
  • H2O: 40uL
  • HindIII: 1uL

Ran half of the reaction on a 0.8% agarose low TAE gel, along with ~500ng of undigested plasmid.

Results:

Gel Loading:

Lane 1 – Hyperladder I (Bioline)

Lane 2 – Undigested construct

Lane 3 – HindIII digestion of construct

We see exactly what we want to see: the construct is fully linearized and runs at the expected size (~5400bp). It runs about half way between the 5000bp and 6000bp markers on the ladder. Will quantify and prepare a dilution curve for the RLP qPCR assay.

Restriction enzyme was selected based on what the construct should look like and HindIII is a single cutter in this instance. Below is an image of the construct, with the insert (AF133090) in green. Of nine common enzymes, four are indicated in the image below as being single cutters. HindIII was selected based on availability in the lab.

PCR – Colony Screening

Eight white colonies were selected for PCR screening to verify that they indeed contain the AF133090 insert, using M13 vector primers. Master mix calcs are here. Using sterile toothpicks, colonies were picked, re-streaked and then used to “inoculate” the PCR reactions. Cycling params were as follows:

1 cycle of:

  • 95C – 10m

40 cycles of:

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

PCR reactions were run on 0.8% TBE agarose gel.

Results:

Gel Loading:

Lane 1 – Hyperladder II (Bioline)

Lane 2 – Colony 1

Lane 3 – Colony 2

Lane 4 – Colony 3

Lane 5 – Colony 4

Lane 6 – Colony 5

Lane 7 – Colony 6

Lane 8 – Colony 7

Lane 9 – Colony 8

Lane 10 – NTC

The prominent bands seen on the gel all run at the expected size (~1600bp). Will select one of these re-streaked colonies for mini prep on Monday.

Cloning – Purified AF133090 PCR Product from earlier today

Purified AF133090 PCR product from earlier today was cloned into pCR2.1 TOPO, using the TOPO TA Cloning Kit (Invitrogen). A full cloning reaction was run. Ligation reaction was incubated for 15mins at RT. Top 10 cells were transformed according to the Rapid Transformation protocol, spread on LB+Amp100 (with X-gal) and incubated O/N at 37C.

PCR – Cloning AF133090 for New RLP Standard Curve

Ran PCR using newly ordered primers (WS_16s_1_F, WS_16s_1501_R) that should amplify the entirety of AF133090 in order to create a new RLP plasmid standard curve for the RLP qPCR assay. Master mix calcs are here. Template used was “Big Reds” fecal DNA extraction by Lisa on 4/3/2012. Sample was run on a 0.8% TBE gel.

Results:

Lane Loading Guide:

Lane 1 – Hyperladder I (Bioline)

Lane 2 – AF133090 PCR

Lane 3 – NTC

Success! The bright band in the Af133090 sample is at exactly 1500bp, as expected. This band was excised and purified using an Ultra-DA column (Millipore). The extensive smearing in this lane is most likely due to the template used, which consists of a DNA extract from abalone feces. This sample likely contains a high amount of DNA, which results in the high molecular weight smearing seen.

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.

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.

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.

qPCR – Fresh RLP Standard Curve (from earlier today) and Lisa’s Abalone Fecal Samples

Ran qPCR using the WSN1F/R primers w/RLP_p probe on the RLP plasmid curve prepped earlier today. Also ran three (Blacks, Big Reds, Round Reds) abalone fecal DNA extracts from Lisa (from 4/3/2012). Master mix calcs are here. Plate layout, cycling params, etc can be found in the qPCR Report (see Results).

Results:

qPCR Data File (CFX96)

qPCR Report (PDF)

Still seeing the same issue of having a separation between two points of the curve greater than 3.32 Cq values (i.e. greater than a 10-fold dilution). Since I’ve been running these samples in the same two rows of the machine during all my testing (rows A & B), I will repeat this with my samples in different rows than I’ve been using previously to help rule out that the CFX96 machine is behaving badly. It is highly, highly unlikely that the machine is at fault, but might as well test it since I can’t come up with any other possible solutions. Will also have Lisa set up a curve from my fresh plasmid prep and we will also perform a run on a different qPCR machine.

20120406 – UPDATE: Carolyn has just remembered that there are multiple, different versions of RLP cloned into a variety of vectors. After discussion with Carolyn and Robyn, Carolyn definitively determined that Lisa and I had not been using the correct clones with the correct RLP insert! The correct vector/insert should be p16RK3-C with the entire AF133090 sequence insert.

 

Repeat of qPCR from earlier today

Repeated the qPCR from earlier today, but placed the samples in different rows than used previously to help rule out that the CFX96 machine is malfunctioning. Master mix calcs are here. Plate layout, cycling params, etc. can be found in the qPCR Report (see Results).

Results:

qPCR Data File (CFX96)

qPCR Report (PDF)

Unfortunately, running these samples in a different location on the qPCR block didn’t alter the results which suggests that the machine is functioning properly. Still seeing the same issue of having a separation between two points of the curve greater than 3.32 Cq values (i.e. greater than a 10-fold dilution). Hopefully having Lisa make and run a curve will add some light to the issue.

I also will try a run using a different polymerase, as this is the only aspect of the reaction that has NOT been tested. Additionally, the problems seem to coincide when we received a new box of Immomix (Bioline). AND, looking at the standard curve, it has become clear to me that the EFFICIENCY of the reaction is bad (~56%) which indicates that the reaction itself is not functioning properly.

20120406 – UPDATE: Carolyn has just remembered that there are multiple, different versions of RLP cloned into a variety of vectors. After discussion with Carolyn and Robyn, Carolyn definitively determined that Lisa and I had not been using the correct clones with the correct RLP insert! The correct vector/insert should be p16RK3-C with the entire AF133090 sequence insert.

Plasmid Curve – RLP Plasmid Size Determination

After looking through Nate’s notebook and aligning the withering syndrome primers and probe to their source sequence (GenBank Accession AF133090; see below) I have discovered a few things:

  1. The vector that was used for cloning was Invitrogen’s PCR 4-TOPO (3956bp). This was found in Nate’s notebook #1 WS qPCR on page 3.

  2. The insert was generated with the primers RA 5-1 and RA 3-6. The resulting fragment size is 158bp. However, Nate’s notebook lists the size as 176bp.

  3. The size of the vector plus the insert should be 4114bp. However, since Nate used the incorrect insert size, he calculated the combine vector+insert size as 4133bp.

  4. Although this doesn’t seem to have much of an effect on the withering syndrome qPCR assay (based on the assay working for years prior to our current issues), the WSN1R primer sequence actually lies partially outside of the cloned region of AF133090. See the alignment below.

Made the dilutions for the RLP standard curve, based on a size of 4114bp. The calculations are here.

20120406 – UPDATE: Carolyn has just remembered that there are multiple, different versions of RLP cloned into a variety of vectors. After discussion with Carolyn and Robyn, Carolyn definitively determined that Lisa and I had not been using the correct clones with the correct RLP insert! The correct vector/insert should be p16RK3-C with the entire AF133090 sequence insert.