Cloning – Purified Clam RLO PCR

Purified PCR product (universal ehrlichia primers) from 20150219 was used for cloning.

Purified PCR volume: 57μL

Purified PCR amount: 75ng (estimated from ladder on gel)

Purified PCR conc: 1.3ng/μL (calculated from numbers above)

The PCR product was ligated using the TA Cloning Kit (Invitrogen).

LIGATION

Ligation reaction:

  • PCR product: 4μL
  • 10x Buffer: 1μL
  • Vector (pCR2.1): 2μL
  • Water: 1μL
  • T4 Ligase: 1μL

Incubate O/N (~18hrs) @ 14C.

Lysogeny broth (LB) plates were made: (100mL of 1x LB) containing 1.5% agar (1.5g), autoclaved, cooled, 500μL of 20mg/mL ampicillin added (50μg/mL final concentration), mixed, and poured.

TRANSFORMATION

40μL of X-gal (40mg/mL) was added to a LB-Amp50 plate, spread and warmed @ 37C.

A vial of OneShot TOP 10 chemically competent cells (Invitrogen) were thawed on ice. 5μL of the ligation reaction was added to the cells, gently mixed and incubated on ice for 5mins. The vial was heat shocked @ 42C for 30s and immediately transferred to ice. The vial of cells was transferred to the LB-Amp50+X-gal plate, spread and incubated O/N at 37C.

 

Sequencing – Coral RLO

Sanger sequencing data from Sonja’s plasmid clones #1, #2, & #3 from 7/29/2013. As of 9/20/2013 Sonja’s notebook is missing, so the full cloning process is not documented. However, I know cloning was done with Invitrogen’s TA Cloning Kit in vector pCR2.1. The three clones were each sequenced with M13 forward and reverse primers. Raw sequence data is here:

20130920_Coral_RLO (Google Drive folder)

  • #1 M13F (039_B10_SJW01-F.ab1)
  • #1 M13R (042_G12_SJW01-R.ab1)
  • #2 M13F (040_A10_SJW02-F.ab1)
  • #2 M13R (043_F12_SJW02-R.ab1)
  • #3 M13F (041_H12_SJW03-F.ab1)
  • #3 M13R (044_E12_SJW03-R.ab1)

Results:

Aligned the sequences to the sequence from which the original primers were designed (Casas et al 2004, Env. Micro). Seqs match nicely, but there are 6 positions (most easily identified in the “Identity” row in the image below) that differ in DQ007350 from our sequencing data of these three clones. The plasmids will be used as a source for qPCR standard curves as well as for in situ hybridization (ISH) probes.

PCR – Coral Rickettsia PCRs

Ran a series of PCRs:

2nd round of nested PCR (from 20130412)

PCR with Sammi’s primers (CAR1F, CAR1R)

Master mix calcs are here.

Template DNA used for the CAR1 primer PCRs were CC28 and PC45 at 5ng/uL. These were provided by Sammi. I do not know what they are or what the naming scheme indicates.

For the 2nd round of nested PCRs, 2uL from the 1st round PCRs were used as template.

Cycling params:

1 cycle:

  • 95C – 2mins

30 cycles:

  • 95C – 1min
  • 65C – 1min, -0.5C/cycle
  • 72C – 3min

1 cycle:

  • 72C

Results:

PCR – Coral Rickettsia nested PCR, outside primers

Ran this PCR since Sonja and Sammi have had some issues with it. Followed reagent concentrations and cycling params from Casas et. al. (2004) Widespread association of a Rickettsiales-Wke bacterium with reef-building corals. Environmental Microbiology. doi:10.1111/J.1462-2920.2004.00647.X

This is the initial PCR of the nested PCR consisting of the “outside primers.”

Primers used were Ricket87F2 and Ricket447R2. Despite the primer names not matching those published in the paper above, I verified that the sequences on the stock tubes matched the sequences listed in the paper.

Master mix calcs are here.

Cycling params:

1 cycle:

  • 95C – 2mins

30 cycles:

  • 95C – 1min
  • 65C – 1min, -0.5C/cycle
  • 72C – 3min

1 cycle:

  • 72C

Template DNA used were CC28 and PC45 at 5ng/uL. These were provided by Sammi. I do not know what they are or what the naming scheme indicates.

Results:

Will post gel image at a later date once the second round of PCRs are run for this nested PCR.

UPDATE: Gel image can be seen above, on 20130415