# Help PCR fungi

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Hi, can you explain the various steps used in this article in steps? For example "and their
relative quantity was estimated by running 5 μl DNA on
1% agarose gel for 25 min. "i think indicates 
electrophesis

Molecular Analysis
Before DNA extraction, root samples were washed from CTAB
buffer and then homogenized in 2-ml Eppendorf tube using
two 3-mm tungsten carbide beads in Mixer Mill MM400 (Retsch
GmbH, Haan, Germany) at 30 Hz for 5 min. The PowerSoil
DNA Isolation Kit (MoBio, Carlsbad, CA, United States)
was used to extract DNA from homogenized root samples
following the manufacturer’s protocols. PCR was carried out
using a mixture of five forward primers ITS3ngsMixTag1-5
(CTAGACTCGTCAHCGATGAAGAACGYRG) in equimolar
concentration and a degenerate reverse primer ITS4ngs
(TCCTSCGCTTATTGATATGC; Tedersoo et al., 2014b). The
ITS4ngs primer was supplemented with unique 10–12 base
pairs long tags per sample (Supplementary Table S1). Tags
were modified from those recommended by Roche (Basel,
Switzerland) to differ by >3 bases, to start only with adenosine
and to comprise similar proportions of adenosine and thymidine
(between 30 and 70%) to equalize their affinities in an adapter
ligation step (Tedersoo et al., 2014b). The PCR mixture comprised
0.6 μl template DNA, 0.5 μl each of the primers (20 μM),
5 μl 5 × HOT FIREPol Blend Master Mix (Solis Biodyne,
Tartu, Estonia), and 13.4 μl double-distilled water. PCR was
carried out in two replicates in the following thermocycling
conditions: an initial 15 min at 95°C, followed by 30 cycles
of 95°C for 30 s, 55°C for 30 s, 72°C for 1 min, and a
final cycle of 10 min at 72°C. PCR products (typically
350–400 bp) from replicate samples were pooled and their
relative quantity was estimated by running 5 μl DNA on
1% agarose gel for 25 min. DNA samples with no visible
bands were re-amplified with 35 cycles and DNA samples
with strong bands were re-amplified with 25 cycles. Both
negative and positive controls were included in PCR and
sequencing runs. PCR products were pooled at approximately
equimolar ratio as determined by gel band strength. Samples
were combined into two libraries that were purified by
FavorPrep™ Gel/PCR Purification Kit (Favorgen-Biotech Corp.,
Austria), following the manufacturer’s instructions. DNA from
each library was quantified using Qubit® 2.0 Fluorometer
(Invitrogen, Life Technologies, CA, United States) and dsDNA
High Sensitivity assay kit (ThermoFisher Scientific, Waltham,
United States). Amplicons were pooled into two libraries and
subjected to adaptor ligation and Illumina MiSeq sequencing
(2 × 300 paired-end) in NERC Biomolecular Analysis Facility
(Liverpool, United Kingdom).


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So after PCR, they took 5 microliter and ran it on a 1% agarose gel (which is as you say gel electrophoresis). Although this does not tell you the amount of DNA in micrograms, the relative quantity (so between the replicates) can be estimated by looking at the gels. They then use the band strength to decide on how many extra cycles of amplification is needed.

Does that make it clear to you or was your question pertaining more of the steps?

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1 hour ago, Dagl1 said:

...

thanks a lot. unfortunately, however, I cannot understand the steps following the electrophoresis. If you give me a summary I am grateful

Edited by Alex007
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40 minutes ago, Alex007 said:

thanks a lot. unfortunately, however, I cannot understand the steps following the electrophoresis. If you give me a summary I am grateful

Could you give me a bit more information regarding what is not clear? How far do you understand this?
There are not that many steps after this, so you might gain a lot by going over what parts you do understand as a learning experience;

After gel electrophoresis we have:
1. DNA samples with no visiblebands were re-amplified with 35 cycles and DNA samples
with strong bands were re-amplified with 25 cycles. Both negative and positive controls were included in PCR and sequencing runs.

2. PCR products were pooled at approximately equimolar ratio as determined by gel band strength.

3. Samples were combined into two libraries that were purified by FavorPrep™ Gel/PCR Purification Kit (Favorgen-Biotech Corp., Austria), following the manufacturer’s instructions.

4. DNA from each library was quantified using Qubit® 2.0 Fluorometer (Invitrogen, Life Technologies, CA, United States) and dsDNA High Sensitivity assay kit (ThermoFisher Scientific, Waltham, United States).

5. Amplicons were pooled into two libraries and subjected to adaptor ligation and Illumina MiSeq sequencing (2 × 300 paired-end) in NERC Biomolecular Analysis Facility (Liverpool, United Kingdom).

I assume this is either homework or you are trying to apply this, in both cases it helps to explain the steps up so far you do understand them, and explain which parts are unclear regarding each following step. This might give you pointers at what to look up yourself. What is your background?

Point 1 and 2 should be clear as they are just using definitions, point 3 has the word library in it, but you could just use collection. Point 4 are technical ways of measuring DNA quantity, point 5 is about Illumina sequencing. You might want to watch a video regarding it if you are unsure how this works, you might also want to look up the difference between single and paired-end sequencing, as they use paired-end sequencing!

There is also this much longer workshop, which goes over everything in much more detail (have not watched all of it):
Edited by Dagl1

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