Restriction Digestions – pCR2.1/RLOv Plasmids

Set up restriction digestions to linearize the pCR2.1/RLOv plasmids in preparation for ISH probes and qPCR standard curves. Used BamHI (NEB), since it doesn’t cut in any of the RLOv sequences and cuts one time in pCR2.1-TOPO (Invitrogen).

PLASMID Vol for 1.5μg (μL) H2O to 40μL
pCR2.1/RLOv_DNA_helicase 21.4 18.6
pCR2.1/RLOv_head_to_tail 11.1 28.9
pCR2.1/RLOv_membrane_gene_1 12.2 27.8
pCR2.1/RLOv_membrane_gene_2 14.3 25.7
pCR2.1/RLOv_tail_fiber 20 20

 

Digestion Master Mix

REAGENT SINGLE REACTION (μL) x 5.5 (μL)
Plasmid 40 NA
10x Buffer 3.1 (NEB) 5 27.5
BamHI (NEB) 1 5.5
H2O 4 22
TOTAL 50 Add 10μL to each tube

Digests were incubated at 37C for 1hr in PTC-200 thermal cycler (MJ Research); no heated lid.

Ran 3μL of undigested plasmid and 10μL of linearized plasmid on 0.8% agarose 1x TBE gel stained w/EtBR.

Results:

Hyperladder I (Bioline)

U = Undigested; Bam = BamHI digest

Besides the funky way this gel ran, the digests look to be complete.

Will quantify remaining linearized plasmids with a dye-based method for accurate quantification and then proceed with the making ISH probes (membrane genes and tail fiber gene) or qPCR standard curves (DNA helicase and head-to-tail).

 

Restriction Digestion – Withering Syndrome Phage ORF25 Plasmid from 20140926

Performed restriction digest using NcoI (NEB) to linearize plasmid for use as qPCR standard curve. Reactions were run for 1hr @ 37C and then heat inactivated @ 65C for 20mins.

Each reaction contained:

pCR2.1/Phage ORF25 (~1ug) – 17uL

10x Buffer 3 – 5uL

NcoI – 1uL

H2O – 27uL

Total: 50uL

After inactivation, 5uL from the reaction was run on a 1% TBE gel to confirm digestion. 5uL of undigested plasmid was run along side the digest.

Results:

Gel Loading Guide:

Lane 1 – Hyperladder I (Bioline)

Lane 2 – pCR2.1/ORF25 (undigested)

Lane 3 – pCR2.1/ORF25 (NcoI)

Vector size (bp): 3929

Insert size (bp): 483

Total size (bp): 4412

The linearized plasmid (which contains a single NcoI recognition site) runs between the 5000 and 4000bp standards, which is what we expect. Will quantify and generate dilution series for use as a qPCR standard curve.

Restriction Digests – Withering Syndrome Plasmds 20131211

Linearized three plasmids for use in WS qPCR standard curve NcoI. Each reaction contained the following:

Plasmid: 5uL

10x Buffer #3 (NEB): 5uL

H2O: 39uL

NcoI (NEB): 1uL

All reactions were incubated @ 37C for 1hr and then heat inactivated @ 65C for 20mins. 25uL of each reaction was run on a gel, along with 5uL of undigested plasmid to confirm digestion.

Results:

Gel Layout

  • Lane 1 – Hyperladder II (Bioline)
  • Lane 2 – pCR2.1/WS-1
  • Lane 3 – pCR2.1/WS-1 NcoI
  • Lane 4 – pCR2.1/WS-2
  • Lane 5 – pCR2.1/WS-2 NcoI
  • Lane 6 – pCR2.1/WS-3
  • Lane 7 – pCR2.1/WS-3 NcoI
  • Lane 8 – No template control

Ran the wrong ladder. However, we see complete digestion of pCR2.1/WS-1 and WS-2. pCR2.1/WS-3 may not be complete, as evidenced by the highest molecular weight band that is present in both the undigested and digested lanes. Although this has no effect on anything, pCR2.1/WS-3 has the insert in the opposite orientation as WS-1 and WS-2, which explains the different digestion pattern. Will create standard curves from all three digestions.

Restriction Digest – Withering Syndrome Clone Plasmid (p16RK7) from 20131106

Performed restriction digest using NcoI. Reactions were run for 1hr @ 37C and then heat inactivated @ 65C for 20mins.

Each reaction contained:

  • Plasmid – 5uL
  • 10x NEB Buffer 3 – 5uL
  • NcoI – 1uL
  • H2O – 39uL

After inactivation, 5uL from each reaction were run on a 1% TBE gel to confirm digestion. 1uL of undigested plasmid was run along side the digest.

Results:

Chloroform Cleanup – EcoRI-digested Withering Syndrome Phage DNA from earlier today

To concentrate the sample, a chloroform cleanup and EtOH precipitation was performed. An equal volume of chloroform was added to the sample (220uL), vortexed for 30s and spun @ 16,000g at 4C for 15mins. The aqueous phase was transferred to a clean tube for EtOH precipitation.

0.1 volumes (16.8uL) of 3M NaOAC (sodium acetate; pH = 5.2) and 2.5 volumes (462uL) of ice cold 100% EtOH were added to the sample and mixed thoroughly. The sample was incubated O/N at -20C. The following day the sample was pelleted by spinning at 16,000g at 4C for 30mins. As expected, there was no visible pellet due to the extremely low quantity of DNA in the sample. The supernatant was discarded, the pellet was gently washed (no mixing/vortexing) with 70% EtOH, and spun @ 16,000g at 4C for 15mins. The supernatant was discarded the pellet was air dried for 15mins. The sample was reconstituted in 10uL of Buffer EB (Qiagen), which is simply 10mM Tris-HCl, and stored at 4C.

Gel Purification – EcoRI-digested pCR2.1 Vector from earlier today

Gel-excised band from earlier today was purified using the Ultra DA-free (Millipore) spin column according to the manufacturer’s protocol. Purified DNA was stored at 4C.

Restriction Digestions – Withering Syndrome Phage DNA and p16RK3

Performed restriction digestions on Phage RLO DNA (isolated on 20121130) and p16RK3 using EcoRI from NEB.

Phage RLO Digest

  • DNA – 192uL
  • 10x EcoRI Buffer – 22uL
  • EcoRI – 4uL
  • H2O – 2uL
  • TOTAL = 220uL

p16RK3 Digest

  • DNA (1ug) – 5.39uL
  • 10x EcoRI Buffer – 5uL
  • EcoRI – 1uL
  • H2O – 38.61uL
  • TOTAL = 50uL

Samples were incubated at 37C for 1hr. The p16RK3 sample was run on a 0.8% TBE gel to confirm digestion and isolate the vector-only band from the sample. The Phage RLO sample was subject to a chloroform cleanup and EtOH precipitation.

Results:

Digestion of Phage RLO DNA was not verified due to the extremely low quantities of DNA. In order to minimize loss of material, we will trust that the digestion was successful if the digestion of p6RK3 was successful.

Gel Loading

Lane 1 – Hyperladder I (Bioline)

Lane 2 – p16RK3 EcoRI

Neglected to run undigested p16RK3. However, the digestion pattern looks correct. The empty vector (pCR2.1; Invitrogen) should be 3931bp and we see the largest molecular weight band of the digest is running at ~4000bp. That band was excised and will be purified for subsequent ligation.

Restriction Digests – Withering Syndrome Clone Plasmids from 20120718

Performed restriction digest on all four clones using NcoI. Reactions were run for 1hr @ 37C and then heat inactivated @ 65C for 20mins.

Each reaction contained:

Plasmid – 5uL

10x Buffer – 5uL

NcoI – 1uL

H2O – 39uL

After inactivation, 5uL from each reaction were run on a 1% TBE gel to confirm digestion. 1uL of undigested plasmid was run along side the corresponding digest.

The four clones are referred to as:

  • pWC8
  • p16RK3
  • p16RK7
  • p18RK7

Results:

Gel Loading (left to right):

Lane 1 – Hyperladder I (Bioline)

Lane 2 – pWC8 (Und.)

Lane 3 – pWC8 (NcoI)

Lane 4 – p16RK3 (Und.)

Lane 5 – p16RK3 (NcoI)

Lane 6 – p16RK7 (Und.)

Lane 7 – p16RK7 (NcoI

Lane 8 – p18RK7 (Und.)

Lane 9 – p18RK7 (NcoI)

Lane 10 – Hyperladder I (Bioline)

All digests are complete. All clones reveal the same restriction digestion pattern, producing two bands: ~2500bp and ~3000bp. The band sizes total ~5500bp, which is in line (5432bp) with the full withering syndrome 16s clone in the pCR2.1 TOPO vector (Invitrogen). Will quant and prepare a dilution series for qPCR.

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.

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.

Restriction Digest – RLP Clone 9 Plasmid from 20120323

Performed a restriction digestion on the mini prep DNA from 20120323 using NcoI (NEB). Digestion was set up as follows:

DNA (652.8ng) – 24uL

Buffer #3 – 2.8uL

H2O – 0.7uL

NcoI – 0.5uL

Reaction was incubated @ 37C for 2hrs and then heat-inactivated for 10mins @ 65C. Plasmid linearization was verified via 0.8% agarose TBE gel. 2uL of undigested plasmid DNA and 2uL of NcoI digested plasmid were each run on the gel.

Results:

Gel Loading

Lane 1 – HyperLadder I (Bioline)

Lane 2 – Undigested mini prep plasmid DNA (2uL, ~54ng)

Lane 3 – NcoI digestion (2uL)

The undigested plasmid runs between 2500bp and 3000bp. The digested plasmid runs between 4000bp and 5000bp. Although I don’t know the size of the insert, this size for the linearized plasmid seems correct, since the empty vector (assumed to be pCR2.1) is 3900bp.

Things look good. Will proceed to quantifying the linearized plasmid and then proceed with making and evaluating a new standard curve for qPCR with the WSN1 primer/probe set.

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.