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 Curve Check – pCR2.1/AF133090

qPCR – Fresh RLP Plasmid Curve from earlier today

Ran qPCR on the new pCR2.1/AF133090 construct I created, in hopes of finally getting a working stand curve for the RLP qPCR assay. Master mix calcs are here. Additionally, three fecal extract DNA samples were run that Lisa was curious about quantifying (these have been run previously). All samples were run in duplicate. Plate layout, cycling params, etc can be found in the qPCR Report (see Results).

Results:

qPCR Data File (CFX96)

qPCR Report (PDF)

 

Plasmid Curve – Linearized pCR2.1/AF133090 from 20120430

Created a fresh dilution of this new construct using Low TE Buffer. Dilution calcs are here.

 

DNA Quantification – Linearized pCR2.1/AF133090 Construct from DATE

Performed DNA quantification using Pico Green and the Tecan plate reader, according to protocol.

Results:

The linearized construct was quantified as having 20.221ng/uL. Raw fluorescence, the mean quantity and plate layout are here. The results from the standard curve for quanting are below.

R2 = 0.99961

y = 503.74x + 221.84

d = 196.97

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.

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.