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[求助]
新人请教一个PCR问题，请多多帮助啦
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这是今天PCR的结果 目的片段是200bp，marker从上至下依次是2000，1000,750,500,300,100 不知道这样的结果算得上是有条带吗？ 不知道这种情况下该怎么改进呢？ 谢谢各位了，在线等！  |
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12楼2011-09-09 13:42:25
wanhscn
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【答案】应助回帖
★ ★ ★ ★ ★
dhd997(金币+5, MolEPI+1): good 2011-09-09 18:06:02
dhd997(金币+5, MolEPI+1): good 2011-09-09 18:06:02
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Troubleshooting discussion is based on the PCR protocol as described in the table below. All reactions are run for 30 cycles. From Yale University （这个是我以前做AS-PCR（SNP）的时候查的，给我不少启示） 1. I get (many) longer unspecific products. What can I do? 扩增出比较长的非特异性产物？ Decrease annealing time减少退火时间 Increase annealing temperature升高退火温度 Decrease extension time减少延伸时间 Decrease extension temperature to 62-68º C降低延伸温度到62-68º C Increase KCl (buffer) concentration to 1.2x-2x, but keep MgCl2 concentration at 1.5-2mM.增加KCl浓度到1.2-2.0倍，但是保持MgCl2浓度在1.5-2.0mM Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant.增加MgCl2浓度到3-4.5 mM，但是保持dNTP浓度不变。 Take less primer减少引物 Take less DNA template减少DNA模板浓度 Take less Taq polymerase少用些Taq酶 If none of the above works: check the primer for repetitive sequences (BLAST align the sequence with the databases) and change the primer(s)如上诉无效果：核对引物的重复序列和换引物 Combine some/all of the above 上诉方法灵活使用 -------------------------------------------------------------------------------- 2. I get (many) shorter unspecific products. What can I do? 扩增出比较短的非特异性产物 Increase annealing temperature升高退火温度 Increase annealing time增加退火时间 Increase extension time增加延伸时间 Increase extension temperature to 74-78º C增加延伸温度到74-78º C Decrease KCl (buffer) concentration to 0.7-0.8x, but keep MgCl2 concentration at 1.5-2mM减少KCl浓度至0.7-0.8倍，但是保持MgCl2浓度在1.5-2mM Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant增加MgCl2浓度到3-4.5 mM但是保持dNTP浓度不变 Take less primer少用引物 Take less DNA template少用模板 Take less Taq polymerase少用酶 If none of the above works: check the primer for repetitive sequences (BLAST align the sequence with the databases) and change the primer(s)同第一问题 Combine some/all of the above -------------------------------------------------------------------------------- 3. Reaction was working before, but now I can't get any product. 反应以前有产物，可是现在不行。 Make sure all PCR ingredients are taken in the reaction (buffer, template, Taq, etc)确保反应所有的成分都存在 Change the dNTP solution (very sensitive to cycles of thawing and freezing, especially in multiplex PCR)更换dNTP溶液 If you just bought new primers, check for their reliability (bad primer synthesis ?)新引物要保持其合成的准确可靠性 Increase primer amount增加引物的量 Increase template amount增加模板的量 Decrease annealing temperature by 6-10º C and check if you get any product. If you don't, check all your PCR ingredients. If you do get products (including unspecific ones) reaction conditions as described above.将退火温度降低6-10º C后核对一下是否有产物，如果没有就确认一下PCR反应的成分是否都存在。如果能获得产物（包括非特异性的产物）然后采取上诉方法。 Combine some/all of the above 4. My PCR product is weak. Is there a way to increase the yield? PCR的产物的量很少，怎样增加产量? -------------------------------------------------------------------------------- Gradually decrease the annealing temperature to the lowest possible.逐渐降低退火温度到最大可能 Increase the amount of PCR primer增加引物的量 Increase the amount of DNA template增加模板的量 Increase the amount of Taq polymerase增加酶的量 Change buffer (KCl) concentration (higher if product is lower than 1000bp or lower if product is higher than 1000bp)改变buffer中KCL的浓度（增加如果产物少于1000bp，减少如果大于1000bp） Add adjuvants. Best, use BSA (0.1 to 0.8 µg/µL final concentration). You can also try 5% (v/v, final concentration) DMSO or glycerol.加入辅佐剂。最好用BSA。或者用5%的二甲基亚砜 Check primer sequences for mismatches and/or increase the primer length by 5 nucleotides核对一下一物种错配的，或者增加5个核苷酸 Combine some/all of the above 5. My two primers have very different melting temperatures (Tm) but I cannot change their locus. What can I do to improve PCR amplification? 两个引物的溶解温度非常不一样但不能改变基因座。如何改进？ -------------------------------------------------------------------------------- An easy solution is to increase the length of the primer with low Tm. If you need to keep the size of the product constant, add a few bases at the 3' end. If size is not a concern, add a few bases at either the 3' or the 5' end of that primer.增加低溶解温度的长度。 [ Last edited by wanhscn on 2011-9-6 at 22:29 ] |
2楼2011-09-06 22:25:14
wanhscn
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3楼2011-09-06 22:30:23
quiller2000
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4楼2011-09-06 22:55:20
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