Sample Prep – Withering Syndrome p18RK7 Plasmid Standard Curve Dilution

The withering syndrome standard curve on my last two qPCRs has been wonky, so I’m making a new curve.

Used the 3e7 dilution of p18RK7 from 20161128 as the “stock” to make serial, 1:10 dilutions (final volume of 1000uL in each tube).

I prepared the dilution series using IDTE (pH=8.0; IDT) Buffer. The original dilution worksheet below was set up by Nate Wight many, many, moons ago.

Dilution calcs (Google Sheet): 20160316 – WS p18RK7 plasmid dilution table

Will test out the new curve dilution shortly.

Sample Prep – Withering Syndrome p18RK7 Plasmid Standard Curve Dilution

Lisa recently ran a qPCR and noticed that the standard curve had drifted quite a bit and was no longer usable, so I’m making more.

Used NcoI-linearized p18RK7 abalone withering syndrome plasmid (from 20120726) to make a fresh dilution series. The plasmid was most recently re-assessed and successfully used for a new standard curve on 20160316. As such, I re-used the dilution calculations from 20160316 (see sheet below).

I prepared the dilution series using IDTE (pH=8.0; IDT) Buffer. The dilution worksheet below was set up by Nate Wight many, many, moons ago.

Dilution calcs (Google Sheet): 20160316 – WS p18RK7 plasmid dilution table

Sample Prep – Withering Syndrome p18RK7 Plasmid Standard Curve Dilution

We are virtually out of the withering syndrome qPCR standard curve plasmid dilutions and I need to send some to Colleen Burge at Univ. of Maryland Baltimore County. As such, I’ll need to make a fresh dilution series and test it out prior to sending to her.

Used NcoI-linearized p18RK7 abalone withering syndrome plasmid (from 20120726) to make a fresh dilution series.

Since it’s been nearly 4yrs since the plasmid was prepared, I decided to re-quantify the DNA using a Qubit3 (Life Technologies) in the Roberts Lab. Used the dsDNA BR Qubit reagents.

SAMPLE CONCENTRATION (ng/μL)
p18RK7 NcoI-linearized 29.0

The result is pretty close to what had been measured 4yrs ago (~26.1ng/μL – difference of  ~10%), so that’s good to see.

I prepared the dilution series using IDTE (pH=8.0; IDT) Buffer. The dilution worksheet below was set up by Nate Wight many, many, moons ago.

Dilution calcs (Google Sheet): 20160316 – WS p18RK7 plasmid dilution table

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).

 

Sanger Sequencing Submission – pCR2.1/Clam RLO clones

After the previous round of sequencing analysis, decided to sequence a couple of additional clones from each of the three groups: 16s, EHR, EUB. Submitted ~500ng (3μL) of clones #2 & #3 (C2 & C3) from each group to GENEWIZ for Sanger sequencing (Order #: 10-291940235). Each clone was sequenced from each direction with M13F (-21) and M13R primers for a total of 12 sequencing reactions:

  1. SW01 16s_C2_01-M13F(-21)
  2. SW02 16s_C2_02-M13R
  3. SW03 16s_C3_01-M13F(-21)
  4. SW04 16s_C3_02-M13R
  5. SW05 EHR_C2_01-M13F(-21)
  6. SW06 EHR_C2_02-M13R
  7. SW07 EHR_C3_01-M13F(-21)
  8. SW08 EHR_C3_02-M13R
  9. SW09 EUB_C2_01-M13F(-21)
  10. SW10 EUB_C2_02-M13R
  11. SW11 EUB_C3_01-M13F(-21)
  12. SW12 EUB_C3_02-M13R

Plasmid Isolation – Withering Syndrome Plasmids

Selected colonies 1, 2 & 3 from the colony screens from 20131205 for plasmid isolation. 5mL of liquid cultures were grown O/N at 37C. 1mL of each culture was mixed with 1mL of sterile 50% glycerol solution and stored @ -80C. 3mL of each culture was used for plasmid isolation. Plasmids were isolated using the Qiaprep Mini Spin Kit (Qiagen) according to protocol. Plasmids were eluted with 50uL of Buffer EB.

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.

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.