To what extent does the 260/230 value affect the efficiency of PCR reactions ? Do the constituents leading to a low 260/230 value (EDTA, guanidine salts and oligosaccharides) inhibit PCR ? I have been getting pretty low values of 260/230 (0.5-1.0) for DNA extracted using Charge Switch Protocol using Magnetic beads and the PCR has been failing. Also, is there any reason why I might be getting low 260/230 values in Charge Switch method ? (I was getting good values using Phenol-Chloroform extraction method)

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    $\begingroup$ I've never heard of the charge switch method, I've only ever used phenol chloroform extraction and isopropanol precipitation. If phenol chloroform worked for you before, why not go back to it? $\endgroup$ – user137 Sep 8 '14 at 22:10
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    $\begingroup$ EDTA would certainly inhibit PCR. $\endgroup$ – canadianer Sep 8 '14 at 22:21
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    $\begingroup$ @user137 charge switch is adjusting the ph to change the charge of the magnetic binding bead, change in charge allows washing and elution $\endgroup$ – rhill45 Sep 9 '14 at 1:44
  • $\begingroup$ I doubt it would matter. 1 ul template in a 50 ul reaction - you've diluted it 1:50. There's nothing left - and you could easily go down to 0.2 ul for 1:250. Also, your enzyme makes a big difference. I would email the enzyme manufacturer and ask them. $\endgroup$ – Superbest Sep 9 '14 at 13:14
  • $\begingroup$ I use a 10ul or 20 ul reaction actually, still I get your point. $\endgroup$ – Anurag Mishra Sep 9 '14 at 15:01

EDTA carbohydrates, phenol all have absorbance near 230 nm. TRIzol reagent is a phenolic solution which absorbs in the UV both at 230 nm and ~270 nm

Guanidine HCL used for most DNA isolations will absorb at ~230 nm also

All these things will seriously inhibit your pcr, (and lower your ratio) especially the denaturing agents like guanadine.

All these DNA kits are different spins of a pretty similar process.

If you have a good steady centrifuge stick to the phenol & ethanol. Be certain you are drying your pellet well, but don't over dry it. I'll hear my pellet at 37 for 3-4 min to get rid of ethanol. Ethanol no good in pcr. If you hate doing ethanol precipitation (which I do) cause of invisible pellets, add a high grade bit of glycogen to your aqueous phase before the precipitation. This will make your pellet visible and is completely inert.

If your cleanup isn't the problem do an annealing and Mg gradient and up your primer quantity.

May want to give me more info like the individual absorbance, pcr conditions, template size etc

Traditionally bead purifications gives really good purity, reason I use it for microarrays. But it's expensive and not necessary for pcr.

Charge switch is just changing the charge of the bead by changing the ph.

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    $\begingroup$ My PCR thermocycler conditions are as follows - Initial Denaturation - 95/30s [ Denaturation - 94/10s, Annealing - 57/60s, Extension - 65/9m30s ] x 35 cycles Final Extension - 65/9m30s I am trying to amplify a 10kb fragment from the mitochondrial genome, using NEB LongAmp Taq Polymerase. I use 10ul reactions - 5ul LongAmp MasterMix , 0.4 + 0.4 Forward and Reverse Primers (final 0.5mM in reaction), 1ul BSA (5mg/ml BSA), 1ul Mg2+ (final 1mM in reaction) (optional), 1ul template (amount 40-80ng) and make up volume with RNAase free water. $\endgroup$ – Anurag Mishra Sep 9 '14 at 6:14
  • $\begingroup$ If 260/230 is a problem, it should inhibit all PCR, but I am not getting results for long fragment amplification specifically. For around 1200 bp using Qiagen Hot Start Kit, I am getting results. $\endgroup$ – Anurag Mishra Sep 9 '14 at 6:18
  • $\begingroup$ Your amplifying a pretty big fragment, your going to need close to perfect conditions. 1200bp and 10kb are pretty different in terms of pcr. Also I would check your protocol of how you are getting the mtDNA, you may need to enrich your template by doing a mito purification. Let me know if this makes sense $\endgroup$ – rhill45 Sep 10 '14 at 18:23
  • $\begingroup$ Also your extension times seem too long that pcr reaction must be taking half the day. What does NEB recommend for the extension? $\endgroup$ – rhill45 Sep 10 '14 at 18:25
  • $\begingroup$ @anurag mishra what's the melt temp of your primers? $\endgroup$ – rhill45 Sep 10 '14 at 18:25

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