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sjtea
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- Ó¦Öú: 1 (Ó×¶ùÔ°)
- ½ð±Ò: 2.5
- Ìû×Ó: 6
- ÔÚÏß: 9Сʱ
- ³æºÅ: 1150532
- ×¢²á: 2010-11-19
- ÐÔ±ð: MM
- רҵ: »ùÒò±í´ïµ÷¿ØÓë±í¹ÛÒÅ´«Ñ§

6Â¥2011-09-07 09:43:32
wanhscn
ľ³æ (Ö°Òµ×÷¼Ò)
ÕÜѧ@²Ý¸ù
- MolEPI: 26
- Ó¦Öú: 53 (³õÖÐÉú)
- ¹ó±ö: 0.87
- ½ð±Ò: 3895.7
- É¢½ð: 877
- ºì»¨: 27
- ɳ·¢: 14
- Ìû×Ó: 3642
- ÔÚÏß: 526.2Сʱ
- ³æºÅ: 1376762
- ×¢²á: 2011-08-22
- ÐÔ±ð: GG
- רҵ: Ö²ÎïÒÅ´«Ñ§
¡¾´ð°¸¡¿Ó¦Öú»ØÌû
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dhd997(½ð±Ò+5, MolEPI+1): good 2011-09-09 18:06:02
dhd997(½ð±Ò+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 £¨Õâ¸öÊÇÎÒÒÔǰ×öAS-PCR£¨SNP£©µÄʱºò²éµÄ£¬¸øÎÒ²»ÉÙÆôʾ£© 1. I get (many) longer unspecific products. What can I do? À©Ôö³ö±È½Ï³¤µÄ·ÇÌØÒìÐÔ²úÎ Decrease annealing time¼õÉÙÍË»ðʱ¼ä Increase annealing temperatureÉý¸ßÍË»ðÎÂ¶È Decrease extension time¼õÉÙÑÓÉìʱ¼ä Decrease extension temperature to 62-68º C½µµÍÑÓÉìζȵ½62-68º C Increase KCl (buffer) concentration to 1.2x-2x, but keep MgCl2 concentration at 1.5-2mM.Ôö¼ÓKClŨ¶Èµ½1.2-2.0±¶£¬µ«ÊDZ£³ÖMgCl2Ũ¶ÈÔÚ1.5-2.0mM Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant.Ôö¼ÓMgCl2Ũ¶Èµ½3-4.5 mM£¬µ«ÊDZ£³ÖdNTPŨ¶È²»±ä¡£ Take less primer¼õÉÙÒýÎï Take less DNA template¼õÉÙDNAÄ£°åŨ¶È Take less Taq polymeraseÉÙÓÃЩTaqø If none of the above works: check the primer for repetitive sequences (BLAST align the sequence with the databases) and change the primer(s)ÈçÉÏËßÎÞЧ¹û£ººË¶ÔÒýÎïµÄÖØ¸´ÐòÁкͻ»ÒýÎï Combine some/all of the above ÉÏËß·½·¨Áé»îʹÓà -------------------------------------------------------------------------------- 2. I get (many) shorter unspecific products. What can I do? À©Ôö³ö±È½Ï¶ÌµÄ·ÇÌØÒìÐÔ²úÎï Increase annealing temperatureÉý¸ßÍË»ðÎÂ¶È Increase annealing timeÔö¼ÓÍË»ðʱ¼ä Increase extension timeÔö¼ÓÑÓÉìʱ¼ä Increase extension temperature to 74-78º CÔö¼ÓÑÓÉìζȵ½74-78º C Decrease KCl (buffer) concentration to 0.7-0.8x, but keep MgCl2 concentration at 1.5-2mM¼õÉÙKClŨ¶ÈÖÁ0.7-0.8±¶£¬µ«ÊDZ£³ÖMgCl2Ũ¶ÈÔÚ1.5-2mM Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constantÔö¼ÓMgCl2Ũ¶Èµ½3-4.5 mMµ«ÊDZ£³ÖdNTPŨ¶È²»±ä Take less primerÉÙÓÃÒýÎï Take less DNA templateÉÙÓÃÄ£°å Take less Taq polymeraseÉÙÓÃø If none of the above works: check the primer for repetitive sequences (BLAST align the sequence with the databases) and change the primer(s)ͬµÚÒ»ÎÊÌâ Combine some/all of the above -------------------------------------------------------------------------------- 3. Reaction was working before, but now I can't get any product. ·´Ó¦ÒÔǰÓвúÎ¿ÉÊÇÏÖÔÚ²»ÐС£ Make sure all PCR ingredients are taken in the reaction (buffer, template, Taq, etc)È·±£·´Ó¦ËùÓеijɷֶ¼´æÔÚ Change the dNTP solution (very sensitive to cycles of thawing and freezing, especially in multiplex PCR)¸ü»»dNTPÈÜÒº If you just bought new primers, check for their reliability (bad primer synthesis ?)ÐÂÒýÎïÒª±£³ÖÆäºÏ³ÉµÄ׼ȷ¿É¿¿ÐÔ Increase primer amountÔö¼ÓÒýÎïµÄÁ¿ Increase template amountÔö¼ÓÄ£°åµÄÁ¿ 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.½«ÍË»ðζȽµµÍ6-10º CºóºË¶ÔÒ»ÏÂÊÇ·ñÓвúÎÈç¹ûûÓоÍÈ·ÈÏÒ»ÏÂPCR·´Ó¦µÄ³É·ÖÊÇ·ñ¶¼´æÔÚ¡£Èç¹ûÄÜ»ñµÃ²úÎ°üÀ¨·ÇÌØÒìÐԵIJúÎȻºó²ÉÈ¡ÉÏËß·½·¨¡£ Combine some/all of the above 4. My PCR product is weak. Is there a way to increase the yield? PCRµÄ²úÎïµÄÁ¿ºÜÉÙ£¬ÔõÑùÔö¼Ó²úÁ¿? -------------------------------------------------------------------------------- Gradually decrease the annealing temperature to the lowest possible.Öð½¥½µµÍÍË»ðζȵ½×î´ó¿ÉÄÜ Increase the amount of PCR primerÔö¼ÓÒýÎïµÄÁ¿ Increase the amount of DNA templateÔö¼ÓÄ£°åµÄÁ¿ Increase the amount of Taq polymeraseÔö¼ÓøµÄÁ¿ Change buffer (KCl) concentration (higher if product is lower than 1000bp or lower if product is higher than 1000bp)¸Ä±äbufferÖÐKCLµÄŨ¶È£¨Ôö¼ÓÈç¹û²úÎïÉÙÓÚ1000bp£¬¼õÉÙÈç¹û´óÓÚ1000bp£© 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.¼ÓÈ븨×ô¼Á¡£×îºÃÓÃBSA¡£»òÕßÓÃ5%µÄ¶þ¼×»ùÑÇí¿ Check primer sequences for mismatches and/or increase the primer length by 5 nucleotidesºË¶ÔÒ»ÏÂÒ»ÎïÖÖ´íÅäµÄ£¬»òÕßÔö¼Ó5¸öºËÜÕËá 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? Á½¸öÒýÎïµÄÈܽâζȷdz£²»Ò»Ñùµ«²»Äܸıä»ùÒò×ù¡£ÈçºÎ¸Ä½ø£¿ -------------------------------------------------------------------------------- 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.Ôö¼ÓµÍÈܽâζȵij¤¶È¡£ [ Last edited by wanhscn on 2011-9-6 at 22:29 ] |

2Â¥2011-09-06 22:25:14
wanhscn
ľ³æ (Ö°Òµ×÷¼Ò)
ÕÜѧ@²Ý¸ù
- MolEPI: 26
- Ó¦Öú: 53 (³õÖÐÉú)
- ¹ó±ö: 0.87
- ½ð±Ò: 3895.7
- É¢½ð: 877
- ºì»¨: 27
- ɳ·¢: 14
- Ìû×Ó: 3642
- ÔÚÏß: 526.2Сʱ
- ³æºÅ: 1376762
- ×¢²á: 2011-08-22
- ÐÔ±ð: GG
- רҵ: Ö²ÎïÒÅ´«Ñ§

3Â¥2011-09-06 22:30:23
quiller2000
ľ³æ (ÖøÃûдÊÖ)
- MolEPI: 1
- Ó¦Öú: 160 (¸ßÖÐÉú)
- ¹ó±ö: 0.114
- ½ð±Ò: 2752.9
- É¢½ð: 50
- ºì»¨: 26
- Ìû×Ó: 1168
- ÔÚÏß: 167.3Сʱ
- ³æºÅ: 895537
- ×¢²á: 2009-11-06
- ÐÔ±ð: GG
- רҵ: ΢ÉúÎï×ÊÔ´Óë·ÖÀàѧ
¡¾´ð°¸¡¿Ó¦Öú»ØÌû
¡ï ¡ï ¡ï ¡ï
wanhscn(½ð±Ò+1): ÊÇµÄ 2011-09-06 23:43:09
dhd997(½ð±Ò+3): good 2011-09-09 18:06:17
wanhscn(½ð±Ò+1): ÊÇµÄ 2011-09-06 23:43:09
dhd997(½ð±Ò+3): good 2011-09-09 18:06:17
|
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4Â¥2011-09-06 22:55:20














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