Hi,

The stability of genomic DNA is higher in TE buffer than water. I always prefer to dilute DNA, primers with TE buffer. However, I prepare TE buffer with lower EDTA concentration. i.e 0.5M to 0.1M.

Gentle pipetting is enough to mix, do not vortex. Please use filtered tips to avoid contamination of pipettes and cross-contamination of samples while serial dilution.

I used to dilute the DNA samples by pipetting. I used to do Pipetting up and down gently and later used to use the diluted DNA sample to check in qPCR. I used to develop a kit so I never faced problem when diluting.

And you know developing a PCR kit requires minimum detection limit of DNA. So I never faced an issue.

But make sure, you don't use the DNA sample with repeated freeze thaw cycle. I never used the sample after 3 freeze thaw cycles.

Best buffer to extract DNA is 1X TE buffer for storage. after extraction if you want to do serial dilution for qPCR than it is good to use water. as the TE buffer contain EDTA which inhibit the reaction mostly at 1pico-femtogram DNA concentration. For serial dilution mixing, finger tapping 8-10 time is the best way or very slight (only touch) vortexing, forms the homogeneous mixture of DNA and very less chance for DNA degradation. most time i preferred the 50 to 200 micro liter volume for serial dilution preparation. repeatability of results in the Real time PCR is also depends on the accuracy of pipetting.

Hi everyone.

Mathew A Beale , Venkata Harsha Vardhan Boddeda , Kirti Wasnik , @Dominique Liger

Thank you all for your valuable advicees.

hope this link is useful

https://pdfs.semanticscholar.org/.../7a40eff2061de6ed4d9fc2c4bb47c...

regards

Hi,

The stability of genomic DNA is higher in TE buffer than water. I always prefer to dilute DNA, primers with TE buffer. However, I prepare TE buffer with lower EDTA concentration. i.e 0.5M to 0.1M.

Gentle pipetting is enough to mix, do not vortex. Please use filtered tips to avoid contamination of pipettes and cross-contamination of samples while serial dilution.

Thank you Hassan Nima

Unfortunately the link does not work for me.

Narayanan Kalyanaraman, Thank you for your guidance.

https://www.researchgate.net/deref/https%3A%2F%2Fpdfs.semanticscholar.org%2F...%2F7a40eff2061de6ed4d9fc2c4bb47c

I have always used nuclease-free water to both store and dilute my DNA samples. I prefer not to vortex, I pipette it gently instead. Never had a problem with this method.

Hiba Riyadh Al-abodi , Rafaela Marocci , Thank you for your replies.

How we do it my lab:

Resuspending Primers (generate upon receiving)

Materials: sterile 1.5 mL Eppendorf tube, p1000 micropipette, Bunsen burner, sterilizing 70 % ethanol, Eppendorf tube rack, vortex, table-top centrifuge

1) Using primer OD, calculate nmole content of primer, and subsequently add water to 100 µM.

1b) If primer consists of more than 100 nmoles, then dilute with water to 500 µM solution; clearly label the non-standard concentration on the outside of the tube. Dilute 1:5, as needed.

2) Vortex primer solution to mix.

3) Spin down tube for approximately 2 sec on table-top centrifuge.

Resuspending Plasmids (generate upon receiving)

Materials: sterile 1.5 mL Eppendorf tube, p1000 micropipette, Bunsen burner, sterilizing 70 % ethanol, Eppendorf tube rack, vortex, table-top centrifuge

1) Using primer OD, calculate nmole content of primer, and subsequently add 20:1 TE buffer to add to final solution 100 µM.

2) DO NOT vortex! Tap and flick solution gently to mix.

3) Spin down tube for approximately 2 sec on table-top centrifuge.

Primers are generally ssNA and plasmids are generally dsNA.

@Christopher Skrodzki , Thank you for sharing your method.