FazaBiotech

You could use the table at the end of this doc to roughly evaluate your sample/Primer concentration after running 3ul of them on a 1.5% Agarose Gel.

Always, put your sample/Primer in 1.5 cc tube, mark it well and seal the tube with enough Parafilm-tape to avoid evaporation. Please make sure that sample will be send to us in appropriate conditions to avoid degradation (i.e. -4 C).

If your samples purified

We need about 30 ul (150 ng/ul) or more, use the table at the end of this doc) of the purified PCR product.

If your samples did not purify:

you may need

PCR purification or

Gel purification

[ to see which kind of purification you may need, please refer to next section]

For PCR purification:

We could do it for you if you send 200 ul of your perfect PCR products based on required standards

(Please refer to Next Section for more info).

For Gel purification:

We could not accept them.

These samples only acceptable if:

The customer accepts responsibility of sequencing result anyway.

On the other hand

The customer should obtain a perfect PCR product.

We have two suggestions for customers in such cases:

Gel purification of PCR products (QIA-gene Kit will give you better results).
Try to remove Smears from your PCR products using two-temperature-PCR (93’ 2Min, 28 × [93’ 30Sec, 72’ 1Min], 72’ 3Min). For template only use 0.3 ul of your original PCR product for 25ul PCR reaction.

To have a good sequencing result, Your PCR product should be purified, whether your interested band is good or not. That means:

Here are some examples of the bands need PCR purification (Only have PrimerDimer as contaminator) or Gel Purification (have smear with other contaminators):

The stock of Primer usually has a 100uM concentration. But in some cases the primers will be degraded after several thaw-freezes. So please check quality of your Primers before sending it for us by running 2-3ul of the primer stock on a 1.5% Agarose gel.

If you have a good primer’s band (i.e. A and B at above), send 8ul of your stock in a 1.5 cc tube and seal it with Parafilm carefully. If you have weak bands you should send us more primer to get better results.

Please note that having PCR product is not a good reason for having a good Primer Stock. You may have band even by a weak primer stock. Therefore please check your primer, as mentioned above, before sends it for us to avoid failed sequencing result. For examples all of the above ran primers (i.e. A, B, C & D) on 1.5% Agarose gel had PCR product but only those sharp ones (i.e. A & B ) had good sequencing results. Our experience showed that many of the bad sequencing results are due to bad quantity of primers.

Roughly Evaluation of PCR Product/ Primer Concentration with Agarose Gel Electrophoresis

----------------------------------------------------------------------------------------------------------------------------------------

Primers must be provided by the customers in distilled water.
Primers must be 18-20 nucleotides in length and with a calculated Tm of at least 50C.

For best results, primers must be designed carefully and a Tm of 55 degrees C.

Try to avoid particularly the potential to form primer, dimers and secondary structures.

We stock the universal primers such as T7, T3, SP6, M13F and M13R

Other primers are generally applied by customers.

Primers must be provided by the customers in distilled water. Primers must be 18-20 nucleotides in length and with a calculated Tm of at least 50C.

For best results, primers must be designed carefully and a Tm of 55 degrees C.

Try to avoid particularly the potential to form primer, dimers and secondary structures.

We stock the universal primers such as T7, T3, SP6, M13F and M13R

Other primers are generally applied by customers.

Primers must be provided by the customers in distilled water.
Primers must be 18-20 nucleotides in length and with a calculated Tm of at least 50C.