[交流] 【求助/交流】请教达人：想找出两个细菌中差异表达的基因，有什么成熟且便宜的方法？

现有两株细菌，暂命名为1和2，其中2对于某种抗逆环境具有更高的抵抗力，现在想找出在这种环境下两个菌株差异表达的基因，不知有什么办法？其中菌1已经完成了基因组测序，菌2未测序，由于经费有限，不太可能采用全基因组芯片方法，请教各位达人，有什么既便宜又比较成熟的方法？不胜感激！

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1楼 2010-12-07 09:21:36

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gastrodia

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qinjysdu(金币+2):谢谢参与
qinjysdu(金币+5):谢谢，该方法应该可行！ 2010-12-08 08:29:19

不知道差减杂交是不是可以

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4楼2010-12-07 23:34:29

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xp198766

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qinjysdu(金币+2):谢谢参与
qinjysdu(金币+2):谢谢，这个方法就怕自己找出的可能的基因没有包含实际的差异基因！ 2010-12-08 08:30:35

可不可以在抗逆条件下提RNA，找些可能的基因做qPCR看看~~~~

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3楼2010-12-07 22:33:42

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wangleisdnu

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qinjysdu(金币+2):谢谢参与
rainwander(金币+2):鼓励积极参与~ 2010-12-08 08:15:07

Non-radioactive RNA fingerprint procedure
First strand cDNA synthesis was performed with 15 mg of total
RNA, 500 pmol oligo(dT)18 and 800U Superscript II reverse
transcriptase (Life Technologies) in a volume of 100ml RT
buffer, following the manufacturer’s instructions. After heat
inactivation of reverse transcriptase and RNase A digestion,
reaction products were purified from excess oligonucleotides
by using a QIAquick kit (Qiagen). cDNA derived from 75 ng of
total RNA was used as template for PCR. Amplification was
performed in 20 ml of PCR buffer (10mM Tris-Cl pH 8.3,
50mM KCl, 1.5mM MgCl2) with 125mM dNTPs (Pharmacia)
and 1mM each primer. For each reaction, two 18-mer
oligonucleotides, of arbitrary sequence and a GC content of
61%, were used. Sequences of all 131 oligonucleotides used
in this screen can be obtained upon request. All PCR
amplifications were performed on an Omni-Gene thermal
cycler (Hybaid). After 2 min of denaturation at 948C, 2 units of
Taq DNA polymerase (Roche) were added (manual ‘hot
start’). After 1 min at 948C, the profile was started with two,
low-stringency cycles (948C for 1.5 min, 508C for 3.5 min and
728C for 3 min) followed by 35–40 high-stringency cycles
(948C for 1 min, 658C for 1 min, and 728C for 1 min), and a final
5 min extension at 728C. Reaction products were separated
on 2% SeaKem LE agarose gels (FMC) for 3.5 h at 6–
7Vcm21 and 88C. Gel and running buffer was 0.5X TBE
(50mM Tris-borate pH 7.9, 1mM Na2-EDTA) containing
5mgml21 ethidium bromide. Bands of interest were excised,
DNA was eluted with a Jetsorb kit (Genomed) and the
amplicons were cloned directly by using a Topo TA cloning kit
(Invitrogen).

这种方法比较的原始，应该比较的便宜

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5楼2010-12-08 04:32:53

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