Wednesday, September 9, 2020

Sequencing primers and conditions

I am on the lookout for properties of sequencing primers for the A. catenella hypervariable D1-D2 domain, and primers for the qPCR analysis targeting this domain. I am also looking for the qPCR cycling conditions.

Garneau et al., 2011 selected primers UScatF (5-AACAGACTTGATTTGCTTGG-3) and UScatR (5-CACAGGAGACTTATCATTCATG-3) and produced a predicted PCR amplicon length of 141 bp.

The final qPCRs were  carried out in 50-l volumes containing:

  1.  10 ul of 1:100 crude lysate (5 to 10 ng of environmental DNA), 
  2. a 300 nM concentration of each primer, 
  3. and a 400 nM concentration of fluorogenic probe. 
  4. 1ul PCR colorless GoTaq Flexi buffer, 
  5. 7 mM Mg2,
  6. 200 M dNTP mix, 
  7. and 2.5 units of GoTaq DNA polymerase

All reagents, samples, and standards were prepared on ice prior to thermal cycling. PCRs and a blank control (no DNA added) were set up in triplicate in 96-well PCR plates sealed with flat strip caps (Bio-Rad; 2239441) and centrifuged briefly to remove bubbles. The qPCR thermal cycling conditions were as follows: 1 cycle of heating at 95°C for 3 min and then 40 cycles of 94°C for 15 s (denaturation), 53.2°C for 30 s (annealing), and 72°C for 30 s (extension). Thermal cycling and real-time data collection at the annealing step were performed using an iCycler iQ realtime PCR detection system (Bio-Rad Laboratories)  

Kamikawa et al., 2007 used primers specific to A. catenella: catF (50-CCTCAGTGAGATTGTAGTGC-30) and catR (GTGAAAGGTAATCAAATGTCC-30) and the

 PCR cycles were carried out using 10 ml volumes that consisted of:

  1. 1 ml of temperate DNA, 
  2. 0.3 mM of each primer pair, 
  3. 0.2 mM of the probe, 
  4. 1 LC FastStart DNA Master Hybridization Probes (containing PCR buffer, dNTP, MgCl2, and Taq polymerase) (Roche Diagnostics GmbH), 
  5. Mg2+ to a final concentration of 3 mM(Roche Diagnostics GmbH), 
  6. and PCR grade water to a final volume of 10 ml (Roche Diagnostics GmbH). 

The cycling conditions were as follows: one cycle at 95 8C for 1 min; 50 cycles at 94 8C for 15 s, 56 8C for 30 s, and 72 8C for 30 s.

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