33 replies to this topic

[tuckern]

100bp sounds too big for primer dimer - your oligos would need to be >50 bases each to get that - looks like a proper pcr product. MgCl2 titration might help, as well as trying different Tms.

[sillybiochemist]

1)  Try the DMSO up to 5%.  Some polymerases are ok with DMSO and others aren't.  You never know until you try.

2)  Try a two step PCR.  There is a paper in Biotechniques about a two step PCR reaction but I don't have the reference with me since I'm at home and it's in lab.  Its worth looking up.  Basically you've got template plus forward primer in one tube and template plus reverse primer in another tube and then do between 1-15 rounds of PCR.  Then combine the contents of the two tubes and do your normal 25 rounds of PCR.  Sometimes this allows the primer to bind to the template effectively and allow you to get some initial transcripts which will then bind to each other when you combine the contents of the two tubes.

3)  If you're not using a polymerase for high GC content, then get one.  It will save you a lot of hassle.  It won't solve your problem but it will help you narrow down things when troubleshooting.  KOD XL polymerase (novagen) isn't sensitive to DMSO up to 5% and it helped me get all of my mutants but not without a lot of tries just because the GC content of primer and template were over 60%.  Some people like the quik change kit by stratagene but I never got it to work, which is really expensive when its $600 a kit for 30 rxn or something like that.  To each his own.

4)  I wouldn't go below 55 deg for an annealing temp.  50 might be fine but below that you're pushing your luck with trying to have a high enough temp to resolve any hairpin structures which are common in high GC rich primers not to mention possibly decreasing the specificity of matching primer and template.

good luck!  it takes a lot of troubleshooting and its hard to get consistent results.

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[siri]

bouttela, on Mar 17 2003, 08:01 AM, said:

Hi everyone, Am new to this so any help would be appreciated. 
I have designed primers with restriction enzyme sites at the 5' and 3' ends respectively and am having trouble getting the desired product.   Template is the gene itself gel purified and keep getting bands at approx 100 bp.  I'm thinking that these are primer dimers...Don't want to have to redesign primers.  Another problem is the Tms are high - 68 . 
Thanks,
New frustrated tech

May be you can try by reducing your primer concentrations.
_siri

[saly]

i design primer but Gene not identification until now from mycology but identified with higher plant, designed primer must the band of gene appeared at 420 BP as higher plant but appeared sharp band at 150-100 BP only and primer-dimer not formed for control or may pale pale band and disappeared with time, this may be specific band or not.  
  
saly
saly4ever@hotmail.com

[Felipillo]

do you have try tochdown pcr

[saly]

Hi,
please help me

i made PCR reaction & RT-pCR, the result was as this :

PCR reaction DNA (sample) give band at 150 bp
PCR reaction DNA(negative control) give band at 150 bp
                       But
RT- PCR RNA (sample) give band at 150 bp
RT- PCR RNA (negative control) no band (clear)

** please, what is the Explain of this situation ?
*** this band was primer- dimer or other thing ( as Original band " spesific band" )
thanks for and sorry for elongation

[saly]

saly, on Feb 24 2009, 05:26 PM, said:

Hi,
please help me

i made PCR reaction & RT-pCR, the result was as this :

PCR reaction DNA (sample) give band at 150 bp
PCR reaction DNA(negative control) give band at 150 bp
                       But
RT- PCR RNA (sample) give band at 150 bp
RT- PCR RNA (negative control) no band (clear)

** please, what is the Explain of this situation ?
*** this band was primer- dimer or other thing ( as Original band " spesific band" )
thanks for and sorry for elongation

saly4ever@hotmail.com

[Adrian K]

siri, on Mar 5 2005, 11:40 AM, said:

bouttela, on Mar 17 2003, 08:01 AM, said:

Hi everyone, Am new to this so any help would be appreciated. 
I have designed primers with restriction enzyme sites at the 5' and 3' ends respectively and am having trouble getting the desired product.   Template is the gene itself gel purified and keep getting bands at approx 100 bp.  I'm thinking that these are primer dimers...Don't want to have to redesign primers.  Another problem is the Tms are high - 68 . 
Thanks,
New frustrated tech

May be you can try by reducing your primer concentrations.
_siri

Exactly as siri had said.

I used to have the same problem of high TMs (67C), and also a high CG template. When I used the 10 times dilution of my primer and my working tm is now drop to 61C. Also, try 5 % DMSO, hotstart polymerase and maybe lower your dNTPs concentration.

[Adrian K]

saly, on Feb 25 2009, 07:35 AM, said:

saly, on Feb 24 2009, 05:26 PM, said:

Hi,
please help me

i made PCR reaction & RT-pCR, the result was as this :

PCR reaction DNA (sample) give band at 150 bp
PCR reaction DNA(negative control) give band at 150 bp
                       But
RT- PCR RNA (sample) give band at 150 bp
RT- PCR RNA (negative control) no band (clear)

** please, what is the Explain of this situation ?
*** this band was primer- dimer or other thing ( as Original band " spesific band" )
thanks for and sorry for elongation

saly4ever@hotmail.com

Hi saly, if I not wrong you might have cross DNA contamination in your PCR reaction, most probably the DNA contamination had gone into the taq polymerase you are using. Try using your friend's taq and see if this happens again.

[Rob Steuart]

i'd agree with giving a hot start PCR a go. If you havent got any hot start taq in your lab just wait until the sample plate reaches 95 before putting your samples on

[neuronzy]

For primer dimer, you can try this: mix the sense and antisense primer and then boil them in 100C for 5min, then immediately put on ice (avoid melting in room temperature). After that add other reagents of PCR reaction.
If the Tm of primer is as high as 68C, I have successful doing a two-step PCR: {98C 10s---68C 1min}×30 cycles.
Hope that help.

[susanna]

when designing primers, you have to check the free energy of these primers, forming homo and heterodimers. If this is to high, dont select them. and design others

greetz

[Maddie]

Two cools    but expensive    instruments to help get rid of primer dimers when everything else has failed (and you don't want to cut gels):
1) Automated DNA Size Selection and Collection from Sage Science
http://www.sagescience.com/
2) Flashgel recovery system
http://www.lonza.com...s/flashgel.html

[Maddie]

Somebody added another instrument from Invitrogen

3) E-Gel® CloneWell SYBR Safe™ gels
http://www.invitroge...xtraction.html?

[Harvey]

Did you try the primer gradients? Primer concentration is an important factor of forming dimers