DNA extracted from complex matrices is often hard to quantify due to the presence of contaminants such as organic compounds. Moreover DNA coming from many matrices is difficult to be amplified, due to the low concentration or to the presence of genomic DNA of different origins or inhibiting compounds.
Save time and money! Check your DNA before the shipment.
We suggest you to use the following protocol, with a highly purified Taq Platinum HiFi (Invitrogen) with no E. coli DNA contaminants and tailed primers.
You can use a different Taq polimerase but it should be free from bacterial DNA and HiFi. You can use not-tailed primers but keep in mind that they will be more efficient than tailed ones.
Tailed primers for 16S rRNA V3-V4 (Takahashi et al. 2014):
Pro341F: 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNBGCASCAG -3′
Pro805R: 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACNVGGGTATCTAATCC -3′
Tailed primers for ITS2 region (White et al. 1990):
|MgSO4 (50 mM)||1|
|dNTPs mix (10 mM)||0,5|
|Primer forward (10 uM)||1|
|Primer reverse (10 uM)||1|
|Genomic DNA (3-10 ng/ul)||5|
The PCR cycle is the following:
1 cycle: 94°C for 1′;
25 cycles: 94°C for 30”, 55°C for 30”, 68°C for 45”;
1 cycle: 68°C for 7′.
Load 5 μl of the resulting PCR on agarose gel 1,5%: the lowest band concentration accepted is 5 ng/ul.
< PCR bands
Using different PCR conditions could give you results that are not reproducible in our lab.
We suggest to test different DNA concentration and send to us the sample providing the best results.