Sanger Sequencing Data – pCR2.1/Clam RLO clones

Received data from yesterday’s sequencing submission for GENEWIZ order: 10-291940235.  Clones from each of the three groups (16s, EHR, EUB) were sequenced (see below).

Raw sequencing data (.ab1) files were stored on: backupordie/Sequencing Data/Sanger/10-291940235_ab1.zip

  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

DNA Quantification & PCR – Ireland Clam S/6/14 #19 DNA

Quantification

Quantified the DNA isolated 20150130 via NanoDrop1000 (Thermo Fisher) for quick assessment of DNA.

Although the NanoDrop1000 overestimates actual yields, this is still interesting because the overall yield of this sample is greater than either of the samples isolated on 20150122, yet had significantly less starting material.

 

PCR

Ran PCRs with the following “universal” primer sets in attempt to amplify a 16s (prokaryote) fragment from the RLO that is present in this sample. Additionally, ran a universal 18s (eukaryote) primer set to verify the presence of any amplifiable DNA in the sample, in case none of the 16s primers work.

  • 16s/23s-F/R
  • 27F, 1492R
  • EHR16D, EHR16R (universal ehrlichia)
  • EUB-A/B
  • 18s EUK 581 F, 18s EUK 1134 R

Master mix calcs are here: 20150203 – cPCR Clam debris DNeasy

All samples were run in duplicate.

Cycling params were:
1 cycle of:

  • 95C – 10mins

40 cycles of:

  • 95C – 15s
  • 50C – 15s
  • 72C – 2mins

Ran samples from yesterday’s PCR out on a 0.8% agarose,  1x TBE gel w/EtBr

Results:

Ladder is Hyperladder I (Bioline)

Well, ironically, the only thing that shows amplification is the no template controls (NTC) in the universal 16s primer set!  The only useful aspect of this is that it demonstrates that the reagents are functional.

The universal 18s primers don’t seem to amplify anything, either.

Tomorrow, I’ll test these primers out on DNA that I know will amplify, instead of these new clam DNA samples.

PCR – Universal 16s in Ireland Clam DNA

In an attempt to identify a potential rickettsia-like organism (RLO) in the clam sample, ran PCRs with various universal 16s primers:

  • 16s/23s-F/R
  • 27F, 1492R
  • EHR16D, EHR16R (universal ehrlichia)
  • EUB-A/B

Template DNA used were the Ireland clam DNA isolated yesterday/today with the Qiagen stool/DNeasy kits, respectively.

Master mix calcs are here: 20150122 – cPCR 20150122 – cPCR Clam Universal 16s

All samples were run in duplicate.

Cycling params were:
1 cycle of:

  • 95C – 10mins

40 cycles of:

  • 95C – 15s
  • 50C – 15s
  • 72C – 2mins

Ran samples from yesterday’s PCR out on a 0.8% agarose, low 1x TAE gel w/EtBr

Results:

No amplification in either sample (stool or DNeasy kit DNA) with any of the four primer sets.  Will discuss with Carolyn how she wants to proceed.


 

PCR – Colony Screens from Withering Syndrome 16s Cloning from yesterday

Selected 10 white colonies for PCR and restreaking. Master mix calcs are here.

Results:

 

Ladder = Hyperladder I (Bioline)

All colonies produced a band of the expected size (~1500bp). Will select three colonies to grow up for plasmid isolation.

Cloning – Withering Syndrome 16s (GenBank AFF133090) from yesterday

Successful amplification of the withering syndrome 16s gene (from p16RK7 [from 20120718 plasmid prep]) will be used to generate a new plasmid that we can utilize to generate a newly functional withering syndrome qPCR plasmid standard curve.

0.5uL of PCR reaction from plasmid prep p16RK7 (from 20120718) was used in a half reaction of the TOPO TA cloning kit (Invitrogen) and then transformed into TOP10 cells, all according to Invitrogen’s protocol. 40uL of cells were plated on LB+Amp50+X-gal plates and incubated O/N at 37C. White colonies (indicating positive transformants) will be PCR screened tomorrow.

PCR – Full Length AFF133090 (Abalone Withering Syndrome 16s)

Ran conventional PCR to amplify the full length abalone withering syndrome 16s product for subsequent cloning. Used four different DNA plasmid preps as template, for comparison purposes. Plasmid preps used were:

  • p16RK7 (from 20120718)
  • p18RK7 (from 20120718)
  • pWC8 (from 20120718)
  • p16RK7 A (from 20131106)

Primers used:

  • WS_16s_1_F
  • WS_16s_1501_R

Master mix calcs are here: 20131203 – cPCR WS Full Length

All reactions were run in duplicate.

Cycling params:

  • 95C – 10m

40 cycles of:

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

Results:

Ran 20uL (of 50uL reaction) of each sample on 0.8% TBE gel.

Lanes (left ro right):

1 – Hyperladder I (Bioline)

2 – p16RK7 (from 20120718)

3 – p16RK7 (from 20120718)

4 – p18RK7 (from 20120718)

5 – p18RK7 (from 20120718)

6 – pWC8 (from 20120718)

7 – pWC8 (from 20120718)

8 – p16RK7 A (from 20131106)

9 – p16RK7 A (from 20131106)

10 – NTC

11 – NTC

Expected a band of ~1500bp. Bands of all samples (except pWC8) run at ~1500bp. pWC8 runs a bit higher and will not be used for cloning, as part of our troubleshooting the failure of our plasmid qPCR standard curve for withering syndrome.

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.