Illumina Custom Sequencing Primer

[tgyuris]

When you want to sequence with custom sequencing primer, as opposed to the seq-primer supplied in the Illumina cluster generation kit, what is the solution??!!

-You just simple replace the tubes with the custom primer?
-What should be the primer concentration?

Let me know if you can help me with this information?
Best,
Tibor
(new member/first posting)

[kmcarr]

When you want to sequence with custom sequencing primer, as opposed to the seq-primer supplied in the Illumina cluster generation kit, what is the solution??!!

-You just simple replace the tubes with the custom primer?
-What should be the primer concentration?

Let me know if you can help me with this information?
Best,
Tibor
(new member/first posting)

Tibor,

You add your custom primer(s) to the tube of Illumina provided primers. Contact your local Illumina Field Application Scientist or Illumina Technical Support for details on concentration, etc.

[kwaraska]

It depends on whether you need both primers or if the "genomic" pool will interfere with your sequencing.

We use 15ul of 100uM primer in 3 ml of HT1 for HiSeq Rapid. We use .6ul of 100uM and 120ul of HT1 for the HiSeq High Output. The instructions for how to do it in MiSeq are in the manual.

If you are going to mix the primers, you use the primer pool instead of HT1.

[windhorse8]

Hi kwaraska,
Are there any unique specifications for the synthesis of custom primers such as the requirement of HPLC/PAGE purification, or inclusion of phosphorothioate bonds in the primers ? My custom primer sizes are 50 and 64 bases respectively.

Best,
windhorse8

[FWOS]

Ussually you'll want to HPLC purify for good measure and I would ensure that your primers meet the following criteria -

Custom primers must:
Anneal to the P5 end of the library (refer to sequences above).
Be used for read 1 ONLY.
Span any initial constant regions.
Be positioned so that 5'-->3' extension will occur using the sequence of interest as the template.
Have properties which match those of Illumina's primer as closely as possible:
Tm = 66°C
33bp
52% GC
Not have a risk of forming any significant 2° structures (i.e. won't stick to itself, form loops, etc.)

That should be a good start. Also make sure you dilute your custom primer in HT1 buffer. If you dilute down in H20 you'll get no data back (especially if loading straight on a cBot. )

Good Luck!