How to get rid of PCR primer dimer - (Mar/17/2003 )
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 on Feb 24 2009, 05:26 PM said:
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
siri on Mar 5 2005, 11:40 AM said:
bouttela on Mar 17 2003, 08:01 AM said:
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.
saly on Feb 25 2009, 07:35 AM said:
saly on Feb 24 2009, 05:26 PM said:
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.
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
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.
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
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/group/en/products_services/products/researchproducts/flashgel.html
Somebody added another instrument from Invitrogen
3) E-Gel® CloneWell SYBR Safe™ gels
http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Nucleic-Acid-Purification-and-Analysis/Nucleic-Acid-Gel-Electrophoresis/nucleic_acid_gel_electrophoresis/DNA-Band-Extraction.html?
Did you try the primer gradients? Primer concentration is an important factor of forming dimers