promotion image of download ymail app
愛麗絲 asked in 科學生物學 · 1 decade ago

關於RT PCR的問題!!













2 Answers

  • 小陳
    Lv 6
    1 decade ago
    Favorite Answer

    這裡提供最常用也最好的Trizol抽取 組織或細胞 RNA方法

    RNA Extraction Procedure:

    1.) Be sure the mortar and pestle, forceps, spatulas and other needed equipment are sterile and chilled on ice if necessary.

    Note: All tissue samples are immediately placed and stored in RNAlater. RNAlater protects the tissue from RNAse activity until it can be homogenized in TRIzol, so it is not necessary to keep everything frozen in liquid nitrogen.

    2.) Remove tissue sample from freezer and allow it to thaw enough such that the tissue can be removed from the RNAlater. Place the tissue into the mortar.

    3.) Using a sterile razor blade, cut the tissue into small pieces.

    4.) Using a spatula, scrape all the cut tissue pieces together in the mortar and place into the 50ml tube containing the TRIzol.

    Note: For larger tissue samples, it may be necessary to use more than 3mL of TRIzol. Add more TRIzol as necessary.

    5.) Using an electric homogenizer with a heavy gauge generator (12mm), shred the tissue fully and completely. If homogenization takes a while, keep the tube on ice to prevent overheating and RNA degradation.

    6.) Once all pieces of tissue have been ground, transfer of the homogenate into each of the two 2ml eppendorf tubes.

    Note: If more than 3mL of TRIzol have been used, split the homogenate into as many 2ml eppendorf tubes as necessary.

    7.) Using an electric homogenizer, homogenize the samples in TRIzol fully using a small gauge generator (5-7mm). Homogenize each sample tube at least 3 times for at least 1 minute each time. Keep the samples on ice in between each round of homogenization.

    Note: It is important to keep the samples cold. Excessive homogenization in small samples (1ml) can overheat the samples and cause RNA degradation.

    For cultures of cells, pellet out of growth media, wash 3 X PBS, and resuspend in RNA Later.

    For cultures of cells (suspended in solution), quantify, pellet the cells, and resuspend in TRIZOL at a volume of 5 x 10^6 cells / 1mL TRIZOL.

    2009-03-25 20:37:29 補充:

    8.) Let the samples stand for 5 minutes at room temperature. Cellular debris and insoluble material may start to pellet at the bottom of the tubes. If you can see cellular debris and insoluble material start to accumulate at the bottom of the tube, continue on to step 9.

    2009-03-25 20:37:49 補充:

    9.) Centrifuge the homogenate at 10,000g for 15 minutes at 4ºC to spin out cellular debris and insoluble material.

    10.) Carefully transfer the supernatant to fresh, RNase free 1.7ml tubes. If there is an oily/fat layer on top, remove this layer carefully before transferring the supernatant.

    2009-03-25 20:38:36 補充:

    11.) To each TRIzol supernatant, add 1ul of Glycogen (20ug/ul stock).

    12.) Add 250ul Chloroform and shake vigorously by hand for 30 seconds. Shake vigorously! If not shaken vigorously for a good 30 seconds, it will reduce RNA yields.

    2009-03-25 20:38:54 補充:

    13.) Let stand at room temperature for 10 minutes. You should start to see an initial phase separation.

    14.) Centrifuge at 10,000g for 20 minutes at 4ºC.

    2009-03-25 20:39:01 補充:

    15.) Transfer the aqueous layer (top, clear layer) to fresh RNase free 1.7ml tubes. Transfer as much as possible without interfering with the middle layer.

    16.) Add an equal volume of isopropanol to each tube.

    2009-03-25 20:39:15 補充:

    17.) Mix well and incubate overnight at -20ºC.

    18.) Centrifuge at 10,000g for 20 minutes at 4ºC.

    19.) Remove the isopropanol carefully from the pellet and add 1ml of cold 75% ethanol.

    20.) Centrifuge at 7500g for 4 minutes at 4ºC.

    2009-03-25 20:39:38 補充:

    21.) Carefully remove the ethanol, being careful not to disturb the pellet.

    22.) Add another 1ml of cold 75% ethanol and centrifuge at 7500g for 4 minutes at 4ºC.

    23.) Carefully remove the ethanol, and let the pellet air dry at room temperature for 5 minutes.

    2009-03-25 20:39:59 補充:

    24.) Dissolve the RNA pellet in each tube in 100ul of RNase free water.

    Note: Keep the RNA in each tube separate through the purification procedure below. The purified RNA will be combined at the end.

    Quantify each sample using Nanodrop. Run 5ul of each sample on Agarose gel.

    2009-03-25 20:40:33 補充:

    RT reaction

    1. Before performing the RT reaction, heat 5 μg of total RNA in a 10μl volume at 65°C for 5 to 10 minutes and then quench on ice.

    2009-03-25 20:40:46 補充:

    2. Set up the following components in a 1.5 ml Eppendorf tube:

    10.0 μl heat denatured RNA

    3.0 μl 10 x PCR buffer

    2.5 μl 10mM dNTPs

    6.0 μl 25mM MgCl2

    1.0 μl random primers (1.8 mg/ml)

    0.5 μl SuperScript II reverse transcriptase

    17.0 μl water

    2009-03-25 20:41:00 補充:

    3. Leave the samples at 25°C for 10 minutes then incubate at 42°C for 1 hour.

    4. Denature the cDNA at 95°C and place on ice.

    2009-03-25 20:41:20 補充:

    PCR reaction

    1. Set up the following components in a 0.5ml PCR tube:

    6.0 μl cDNA product

    1.5 μl 10 x PCR buffer

    0.2 μl Taq polymerase

    0.5 μl primer 1 (1.0 mg/ml)

    0.5 μl primer 2 (1.0 mg/ml)

    10.3 μl water

    2009-03-25 20:41:27 補充:

    2. Perform PCR with 30 cycles of denaturation: 30 seconds at 95°C; annealing: 45 seconds at 60°C; and extension 60 seconds at 72°C.

    2009-03-25 20:41:39 補充:

    至於設計primer, 可以到 primer 3 plus 的網頁( 只要把你的 template DNA 序列貼入, 再輸入你要的 primer 條件, Tm值, GC%, 等等, 就可以設計出你要的 primer 了~~有不懂再問我~~

    Source(s): 自己實驗經驗
    • Commenter avatarLogin to reply the answers
  • 6 years ago



    • Commenter avatarLogin to reply the answers
Still have questions? Get your answers by asking now.