Different tissue extraction methods are suitable for RNA

(2020年05月08日)

When doing RealTimePCR, cryo rack parameters required for primer design for a pair of primers used in the SYBRGreenI / EvaGreen method and primers for general PCR are different. DNaseI10ul. RNA reverse transcription to cDNA Reverse transcription program (taking MBI's M-MLV as an example) Component addition (20ul system) Addition (40ul system) Template (RNA ) 0.0ul4. 4.5ul ddH2O11. 2. Since most eukaryotic mRNAs have a 3 'Poly (A +) tail, this primer is paired with them, and only mRNA can be transcribed. Preparation of gel electrophoresis gel: 1) Weigh 0. If two specific primers are used in the PCR reaction, the synthesis of the * strand can be performed with the mRNA3inal zui Proximity paired primers start. It can be verified by electrophoresis. We often add 200ul DEPC water.加 Addition amount of components 2mes;

PCRTaqMix10ul 10uMPrimerFW0. Add water to each tube to 25ul. RNase Inhibitor 4ul. For primers, you must have the mental preparation to pick out one or two useful primers from a large number of primers-it is very difficult to find suitable primers. Add 1/10 volume of NaAC (3M) (about 25ul) and pre-cooled equal volume of isopropanol (about 280ul), and let stand at -20 ° C for 20min. RNA extraction (see

RNA extraction and reverse transcription for details) Different tissue extraction methods are suitable for RNA extraction. 95 ℃ 5min; 95 ℃ 15s, 60 ℃ 35s, 40cycles; 72 ℃ 5min; 4 ℃ pause. 2. During the extraction of total RNA, take care to avoid mRNA breakage; take 2ug for RNA formaldehyde denaturing gel electrophoresis detection, if there is DNA contamination, digest with DNaseI (because RNA is easily degraded during processing, it is recommended in the system Add the right amount of RNase inhibitor). After 5 min pre-denaturation at 95 ° C, 15s at 95 35s at 65 ° C

(fluorescence detection), 40 cycles. Digest the DNA with DNaseI. Expression differences between differences.0ul2.0ul, mix well and place in a SLAN fluorescence quantitative PCR instrument.特别 Pay special attention to avoid the presence of primer dimers and non-specific amplification. The volume is too small, which is not conducive to extraction. There are three possibilities for double peaks to appear in the dissolution curve: Primer peaks. In gene expression regulation studies, some housekeeping genes are used to standardize to correct for differences caused by different initial concentrations of samples
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