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In reference to low tm


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#1 seaner

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Posted 07 April 2003 - 08:44 AM

In the post about RNA contamination below the responder mentioned something about low tm. I too am having smearing on a dna gel. I am trying to amplify cDNA from psport vector using m13puc forward and reverse. The library is stored as a glycerol stock. In the past we would amplify directly from glycerol stock, and get no smearing, we are in the process of swithcing our protocol to amplify from an overnight culture instead of directly from glycerol. I haven't been able to get rid of a smearing and or non specific amplification. I use plantnium taq and with the glycerol protocol we did an intial denaturization of 10 min. I assume this isn't needed if amplfying from culture. I haven't been able to get rid of a smearing and or non specific amplification.I am going to try, today, several things including: lower intial denaturization step, less extension time(from :45 to :30, although :45 isn't that long anyhow), Increase anneal time temp from 63 to 65, Increase anneal time. Anyone else out there have any ideas.

#2 yossi.b

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Posted 07 April 2003 - 10:25 AM

hi seaner below are the reasons for PCR smearing
1. check your primer TM.(while smearing - increase).
2. over loading sample.(load less dna).
3. put the reaction tube with out the Taq in 95oC for 1 min and then add the taq and continue the PCR reaction. this step do not allow the taq to work while the temperature is increasing to 95oC for the first cycle.( it is importent just to the first step).
4. RNA contamination in your mini prep - the band is amplified but u can not see it because of the RNA - use RNAse.
5. less importent but try to load the sample with out the PCR oil.
6. check your primer if they anneal with other places in the vector.
7. i'm sorry to mantiaed this but check your PCR program again be sure all the steps are o.k.
8. it is very importent while smearing to use very low amount of the templae (try ng, pico gr , and even ato gr).
9. the most importent step is to kiss the tube before the reaction.
good luck yossi

#3 seaner

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Posted 08 April 2003 - 07:03 AM

Thanks for your thoughts




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