Dear all,
I have done PCR by using KOD FX with two step method ( 5 cycle with Tm 50 and 35 cycle with Tm 55, and elongation time 1.30 minute with size 1356bp, then I got single band without smear, after that I continued purification. because the amount of dna is less than I wanted, so continue pcr again by using my dna purification as a template and KOD plus reagent. in KOD plus using two step method (5 cycle with Tm 50 and 35 cycle with Tm 55, and elongation time 3 minute) but my result in KOD plus I got smear the yield my pcr product was low. then I try changed the condition pcr using one step with TM 55, but I still got smear. and the yield my pcr product was low.
please give me another suggestion about that..,
best regards,
dewi yuliana
1,Use Nested primers at second PCR.
2,Add the temp as the KOD kit required--- how many temp did you add.
3,KOD,1300bp, 1.5 min ---is easy to PCR for sequence or gene clone, why you use two steps? you should optimize the PCR (temp, primer, TM……)
4,elongation time 3 minutes is not good------1.5 min is enough.
5.dna purification with gel extraction？
- Like Hyde I think Nested PCR - using internal primers or hemi nested PCR - using 1 x internal primer and 1 x original PCR primary - will increase you r specificity and reduce your smearing
- In addition I also agree with Hyde that keeping your extension time @ 1.5min will minimise smearing due to non specific reactions
- Use a maximum of 1:100 aliquot of your primary PCR in your secondary PCR
- In that regard you could try titrating down your primary template from 1:100 to 1:1000 and if necessary add an extra 5 cycles to your secondary PCR
- I also think that for your secondary PCR to maximise stringency you should perhaps omit the less stringent 5 cycle starting reaction and just keep your Tm @ 55C; if necessary adding 5 more cycles to ensure sufficient yield
- Better still try touch down PCR from 60C to 55C as described in the protocol attached and below:
- http://bitesizebio.com/2203/touchdown-pcr-a-primer-and-some-tips/
- However, given that your samples look slightly degraded as alluded to by Hyde I am wondering if part of the issue at least could be DNA degradation limiting the amount of product from primary PCR
- How are you purifying your DNA ? Are you using fresh molecular grade water ? Are you treating your area where PCR is performed with DNAZAP in cluding your Gilsons ?
- By good technique and perhaps touch PCR rather than standard PCR you might be able to generate enough amplicon from primary PCR to avoid secondary PCR which always increases your chances of non specific products
- I can assist you with tips for DNA extraction and/or TD PCR if you so wish
dear all,
thanks very much for your attention. I used two step because at the first time PCR by using KOD FX I got unspecific band, then I tried two step finally I got single band, I continue gel extraction for purification my pcr product, then the result for my gel extraction I used as a template using KOD plus.
I used elongation time 3 minute for kod plus because used kod plus elongation time became 500bp/minute.
I wonder about the yield amount of pcr product with kod plus (second PCR) was low.
how about touch down pcr and nested primer? it can increase yield amount my PCR product?
It is the first time I got yield amount was low after using kod plus and result gel extraction as a template. that why I confused about that.
best regards
dewi yuliana
Hi Dewi
regarding good yield of your 1.5kb product from the first round of amplification,
- is there any reason you are not using a 3 min extension in this phase ?
- Also have you tried a gradient with your current primers where you are having problems: You could try from Tm-5C to Tm
- You might find that 35 cycles with a Tm of 50-55C actually gives you enough specific product without having to use 50C and then go up to 55C: You should be able to use 1 x annealing temp which normally approximates to Tm-2C of your primers (or your lowest Tm primer if Tm differ by > 2C)
- Also to improve yield from primary PCR try adding 5% DMSO to your PCR mix; that is 1ul of DMSO in a 20ul PCR reaction mix
- In addition, you could try increasing the concentration of your primers to 20uM (20pmoles/ul) if you currently are adding 1ul of your primers at 10uM = 10p moles per ul to your PCR reaction. In other words is you currently use 1ul @ 5uM or 10uM try using 2-3 ul of each primer
- Finally, to max efficiency of your PCR if you are not already doing so try a single step denaturation cycle of 96C for 5 min: This will activate your Taq (if hot start) but will also ensure that all of your DNA is single stranded at PCR commencement and thus is primed efficiently (as well as specifically) from cycle 1 of 35
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