This article covers the most common causes that can lead to an unsuccessful extraction with non flower matrices.
1. The SCCG control is not freshly prepared
It's very important to make a fresh dilution of your SCCG positive control before performing the non flower extractions. If previously diluted controls are stored at 4C or -20C and re-used the DNA can degrade quickly causing a decrease of available DNA to amplify during the qPCR. This will result in late or non existent HEX signal in your qPCR.
2. The SCCG control is not diluted properly
To make the 1:5,000 dilution of SCCG, 2 subsequent dilutions have to be performed. in both instances only 1 uL is transferred. Pipetting 1 uL can be difficult and it's important to ensure it's actually in your pipette tip. I suggest visually confirming that the 1 uL is in your tip after aspirating it out of the stock tube. Do the same for your second dilution. When adding the 1 uL to the water in your 1.5 mL tube you've labeled for your dilutions, stick the tip into the existing water, dispense the 1 uL into the water and pipette up and down a few times to ensure it has been fully dispensed. Cap the tube and vortex well to mix, then quick spin the tube to bring all of the liquid back to the bottom of the tube. Repeat this process for the second dilution.
3. The SCCG control is not added to the sample
This is straightforward, don't forget to add the diluted SCCG to each sample after removing a portion post lysis.
4. Chloroform was aspirated with the sample
After the chloroform addition and centrifugation, some of the chloroform might be aspirated with the 100 uL of sample. Matrix type can vary the difficulty of removing 100 uL of the supernatant after the centrifugation step. Some difficult matrices include gummy products or a capsules.
5. The sample is not fully vortexed after addition of chloroform
It is important to vortex the sample and chloroform thoroughly so that the solution turns completely white.
6. The sample is not spun fast enough after the chloroform addition
When processing difficult matrices such as gummy products or capsules, a high speed centrifuge will be necessary to better separate the phases. You'll need a centrifuge that will spin 1.5 mL tubes up to or close to speeds of 14,000 rpm.