7 replies to this topic

[Mad researcher]

i have 12 samples to run PCR for. The reaction volume for each should be 25uL
Should i make 12 different master mix or can i make one master mix including dNTP, Taq and Buffer and add H2O to the PCR tubes containing DNA + Primers?

[Trof]

If you have same primers for all, mix everything except template (DNA) pipett to wells and add template.
If you have different  primers mix, everything except primers and template and add separately.

When making mix, make 10% more of the mix than is the overal volume required (i.e. you need 12 reactions, calculate mix for 13 reactions), you will lose some volume due to pipetting. The 10% is for guidance only, you may need less (or rarely more) depending on the accuracy of your pipetts and pipetting.

[hobglobin]

One master mix for 13 samples (I use one extra every ten samples for pipetting error)....and I'd add everything to the mix except the DNA (except you work with different primers). (an it's not a good idea to add Taq to 10x buffer without dilution, therefore ddwater in mastermix....)

And you were faster, trof...anyway will not delete this

Edited by hobglobin, 26 September 2012 - 07:08 AM.

[why]

Why we can't add Taq to 10x buffer directly? What will happen then?

Thank you.

[Mad researcher]

Trof, on 26 September 2012 - 07:03 AM, said:

If you have same primers for all, mix everything except template (DNA) pipett to wells and add template.
If you have different  primers mix, everything except primers and template and add separately.

When making mix, make 10% more of the mix than is the overal volume required (i.e. you need 12 reactions, calculate mix for 13 reactions), you will lose some volume due to pipetting. The 10% is for guidance only, you may need less (or rarely more) depending on the accuracy of your pipetts and pipetting.

Trof, on 26 September 2012 - 07:03 AM, said:

If you have same primers for all, mix everything except template (DNA) pipett to wells and add template.
If you have different  primers mix, everything except primers and template and add separately.

When making mix, make 10% more of the mix than is the overal volume required (i.e. you need 12 reactions, calculate mix for 13 reactions), you will lose some volume due to pipetting. The 10% is for guidance only, you may need less (or rarely more) depending on the accuracy of your pipetts and pipetting.

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I prepare a single master mix with dNTP + buffer keep it on ice,
i add primers into the master mix
i add water to different PCR vials accordingly along with DNA
now i add the TAQ into the MM and distribute it to the PCR vials.

[bob1]

why, on 07 October 2012 - 06:14 AM, said:

Why we can't add Taq to 10x buffer directly? What will happen then?

Thank you.

Salt concentration may affect the protein causing it to precipitate or denature.

[prabhubct]

bob1, on 07 October 2012 - 12:26 PM, said:

why, on 07 October 2012 - 06:14 AM, said:

Why we can't add Taq to 10x buffer directly? What will happen then?

Thank you.

Salt concentration may affect the protein causing it to precipitate or denature.

In ready to use Master mix. the Taq is added in buffer. why can't it precipitate/ denature?

[phage434]

In a pre-made master mix, the buffer is diluted to 1x, and the Taq is relatively stable.  In 10x buffer, there could be issues with enzyme stabilty.  Having said that, I believe Taq is so robust an enzyme that this would not happen, but it is good practice to use care in enzyme handling.