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RNA Isolation from Synechocystis
集胞藻细胞RNA的提取   

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参见作者原研究论文

本实验方案简略版
Journal of Photochemistry and Photobiology B: Biology
Aug 2014

Abstract

The protocol describes the procedure of total RNA isolation from cells of the cyanobacterium Synechocystis sp. PCC 6803. This protocol is also applicable to Synechococcus elongatus PCC 7942 and PCC 6301, Thermosynechococcus vulcanus, and other unicellular and filamentous species of cyanobacteria that do not have thick polysaccharide-containing outer layers. For the latter, Trizol-containing protocols should be adapted. The yield of RNA depends on optical density of cyanobacterial culture and may reach up to 10-20 µg of total RNA per 1 ml of cell culture. RNA isolated by this method can be used for Northern blot hybridization, RT-qPCR, microarrays and Next Generation Sequencing.

Keywords: Synechocystis (集胞藻), Cyanobacteria (蓝藻), RNA isolation (RNA的分离), RNA quality (RNA的质量), Hot phenol (热酚)

Materials and Reagents

  1. Cyanobacterial culture (50 ml) (OD750 ~ 2) grown in BG11 medium (Rippka, 1988)
  2. 95% ethanol for molecular biology (keep at -20 °C)
  3. 70% ethanol for molecular biology (30 ml of RNAse free water and 70 ml 95% ethanol, keep at -20 °C)
  4. 70% ethanol for sterilization
  5. Phenol BioUltra (for molecular biology, TE-saturated, ~73%) (Sigma-Aldrich, catalog number: 77607 ) (keep at 4 °C)
  6. 1 M Tris-HCl (Life Technologies, catalog number: 15568-025 )
  7. 0.5 M EDTA-Na2 (pH 8.0) (Life Technologies, catalog number: AM9260G )
  8. RNase-free autoclaved water
  9. Chloroform (Sigma-Aldrich, catalog number: C2432 ) (keep at 4 °C)
  10. 10 M lithium chloride solution (Fluka, catalog number: 83268 ) (chill on ice before use)
  11. RNAse-free DNAse
  12. Agarose for molecular biology (Sigma-Aldrich, catalog number: A9539 )
  13. TAE electrophoresis buffer (Sigma-Aldrich, catalog number: T9650 )
  14. RNA ladder (Life Technologies, InvitrogenTM, catalog number 15620-016 )
  15. TE buffer (see Recipes)
  16. 50/100 TE buffer (see Recipes)
  17. 10 M LiCl solution in water (see Recipes)
  18. Cell fix solution (see Recipes)

Equipment

  1. Saran wrap (any type)
  2. Autoclave bag (Sigma-Aldrich, catalog number: Z692212 )
  3. Safe-Lock tubes (2.0 ml) (Eppendorf, catalog number: 0030 120.094 )
  4. 50 ml plastic conical polypropylene tubes (sterile, nuclease-free, nonpyrogenic, autoclavable) (Life Technologies, catalog number: AM12501 or Corning, Falcon )
  5. Autoclave
  6. Refrigerated centrifuge with bucket rotor that fits 50 ml conical tubes (Eppendorf, model: 5804 R and Swing-bucket rotor A-4-44 with 50 ml Falcon adapters)
  7. Refrigerated microcentrifuge preset to 4 °C (Eppendorf, model: 5415 R )
  8. Water bath preset to 65 °C
  9. Subzero refrigerators: -20 °C and -70 °C
  10. Fume hood
  11. Ice bath
  12. Multi tube automated vortex (for example, Micro tube mixer MT-400, Tomy Digital Biology)
  13. UV-spectrophotometer (preferably, Nanodrop)
  14. Agarose gel electrophoresis equipment (VWR Mini Gel II Complete Horizontal Electrophoresis System, catalog number: 95043-650)
    Note: Workplace, pipettes, gloves etc. should be cleaned with 70% ethanol before RNA isolation procedures. Conical tubes, Eppendorf tubes, pipette tips should be sterilized by autoclaving in autoclave bags before use.

Procedure

  1. Probes fixation
    1. Prepare 25 ml portions of cell fix solution in 50 ml plastic conical polypropylene tubes. Keep on ice. Pour 25 ml of cyanobacterial cell culture into 25 ml of ice-cold cell fix solution (Figure 1.1). Stir well and proceed to centrifugation or store samples at -20 °C up to 1 month. For 50-ml of cyanobacterial cultures, two 50 ml tubes should be used. Alternatively, 100-200 ml centrifuge bottles resistant to phenol and ethanol may be used.
    2. Harvest fixed cells by centrifugation at 3,000 x g for 5-10 min at 4 °C (Figure 1.2).
    3. Discard cell fix solution leaving 0,5-1 ml of solution to resuspend the pellet. Resuspend the pellet by vortexing or pipetting.
    4. Transfer a suspension into a sterile 2 ml Eppendorf tube. If 50 ml tubes are used for cell fixation and harvesting, combine two probes from 25 ml tubes in one 2 ml Eppendorf tube.
    5. Centrifuge at 3,000-5,000 x g, 4 °C for 3-5 min and completely remove supernatant (Figure 1.3). Leave pelleted cells on ice.
      Note: Probes fixation is applied to kill cells immediately and to avoid possible degradation of RNA. This is extremely important when temperature-dependent changes in specific mRNAs are under study.


      Figure 1. Steps of RNA extraction. 1. Fixation in Cell Fix Solution. 2. Fixed cells after centrifugation. 3. Cell pellet at the bottom of the tube. 4. Cells resuspended in 500 µL of TE-50/100 5. Cell suspension after addition of 1ml of phenol. 6. Cell suspension after vortexing. 7. Incubation in 65°C water bath. 8. Four phases after centrifugation (from bottom to top): blue pellet (if it is green, hot phenol cell lysis was not complete; in this case resuspend again and start from step A4); dark green phenol phase; protein interphase (might be invisible); pink TE-50/100 phase with nucleic acids. 9. Carefully transfer upper TE-50/100 phase into new tube. Use yellow tip. 10. Do not take interphase and phenol phase into the tip. 11. Addition of 300 µl chloroform and 300 µl phenol to TE-50/100-fraction. 12. Homogenous white colored emulsion after vortexing. 13. Three phases after centrifugation (from bottom to top): phenol/chloroform (lower phase); protein interphase; pink TE-50/100 phase (upper phase). This phase should be transferred into a fresh 1.5 ml Eppendorf tube. 14. Repeat steps A9-13 until interphase disappears.

  2. Cell lysis by hot phenol
    1. Resuspend the pellet in 500 µl of sterile ice-cold 50/100 TE-buffer by vortexing or pipetting (Figure 1.4).
    2. Add 1 ml of phenol, vortex and incubate in a water bath preheated at 65 °C for 10 min (Figure 1.5-1.7). Regularly shake probes (3-4 times during 10 min of incubation) to prevent phenol phase separation.
    3. Centrifuge samples at top speed (16,000 x g) at 15-20 °C for 10 min. The pellet in the bottom phase should be blue-colored meaning cell lysis is complete (Figure 1.8).
    4. Transfer upper aqueous phase to a new Eppendorf tube (Figure 1.9-1.10).

  3. Purification of nucleic acids by phenol/chloroform
    1. Add 300 µl of TE-saturated phenol and 300 µl of chloroform to the probe (collected aqueous phase) (Figure 1.11).
    2. Vortex for 1-2 min and centrifuge at 16,000 x g, 4 °C for 5 min (Figure 1.12-1.13). For multiple samples apply multi tube automated vortex (for example, Micro tube mixer MT-400).
    3. Transfer upper aqueous phase to a new tube.
    4. Repeat this procedure 2-3 times until the interphase (a whitish, denatured protein-containing layer between phenol phase and aqueous phase that contains nucleic acid) disappears completely (Figure 1.14).

  4. Precipitation of nucleic acids
    1. Add 2-3 volumes of 95% ethanol prechilled at -20-30 °C to a final aqueous phase (~ 1,5 ml of ethanol to ~ 0.5 ml of a probe), stir well and incubate at -20 °C from 2-3 h to overnight.
    2. At this stage a probe can be stored for several months under ethanol at -20 °C.
    3. Centrifuge at top speed, 4 °C for 10 min, carefully discard liquid.
    4. Wash the pellet of nucleic acids with 1 ml of 70% cold ethanol by intensive vortexing.
    5. Centrifuge at top speed, 4 °C for 5-15 min and completely remove ethanol by aspiration.
    6. It is important to carry on all operations with RNA on ice.

  5. Separation of RNA from DNA by LiCl precipitation
    1. Add 800 µl of 50/100 TE buffer to the pellet. Incubate for 15 min on ice.
    2. Vortex probe 3-4 times to dissolve the pellet completely.
    3. Add 200 µl of 10 M LiCl solution and mix by vortexing. Leave probes on ice for 2 h.
    4. Centrifuge at top speed, 4 °C, for 15-20 min.
    5. Carefully remove supernatant by aspiration.
    6. Wash the RNA preparation by adding 1 ml of ice-cold 70% ethanol and intensive vortexing of the pellet. Centrifuge at top speed, 4 °C, for 15 min.
    7. Repeat 70% ethanol washing.
    8. Carefully remove all ethanol and air-dry the probe(s) at room temperature in a tube rack covered by Saran Wrap. If vacuum drier is used, please, take care not to overdry the pellet, because it will become impossible to solubilize it. Dissolve RNA in 50-100 µl of RNAse-free water.

  6. Evaluation of RNA quantity and quality
    1. Take 1-2 µl of RNA and check its concentration with Nanodrop spectrophotometer.
    2. LiCl does not allow to obtain RNA that are completely free from DNA, but the quality of RNA is usually enough for nothern blot analysis. If necessary, purify RNA sample(s) additionally with DNAse according to the manufacturer instructions.
    3. Check RNA quality by agarose gel electrophoresis. Electrophoretically resolved total cyanobacterial RNA is visible as 3 major bands that correspond to 23S, 16S and 5S RNAs. Smear on lines indicate RNA was degraded during steps of isolation (Figure 2).


      Figure 2. Total RNA samples isolated from Synechocystis sp. PCC 6803 at 1 µg per lane were separated by electrophoresis in 1% non-denaturing agarose gel and stained with ethidium bromide. Typically 3 major (or more) bands corresponded to 23S, 16S and 5S RNAs should be seen. Lanes 1-3 represent isolated total RNA of good quality, and lanes 4-6 with smeared bands demonstrate low quality partly degraded RNA. For northern blotting, RNA should be separated in denaturing formaldehyde-containing 1.2% agarose gel, and transferred onto nitrocellulose or nylon membrane (lanes 7, 8). It is recommended to run two extra lanes of RNA sample (lane 7) and RNA ladder (lane 8), which will be cut off and stained with methylene blue to determine the size and quality of RNA after blotting.

Notes

Solution of LiCl contains DNA, and it’s important to remove supernatant completely. Also DNA in LiCl solution can be precipitated by 2 volumes of ethanol or 0.8 volumes of 2-isopropyl alcohol (Lab Scan, C19C11X).

Recipes

  1. TE buffer
    10 mM Tris-HCl (pH 7.5-8.0)
    1 mM EDTA-Na2
  2. 50/100 TE buffer
    50 mM Tris-HCl (pH 7.5-8.0)
    100 mM EDTA
    After preparation, this buffer should be sterilized by autoclaving at 121 °C, 1.5 atm., 15 min
  3. 10 M LiCl solution in water
    Note: It should be autoclaved, aliquoted in sterile conditions, and stored sterile.
  4. Cell fix solution
    0.5% phenol in 95% ethanol
    25-ml aliquots in autoclaved 50-ml tubes should be stored at -20 °C

Acknowledgments

This work was supported by a grant from Russian Science Foundation No. 14-24-00020 to D.A.L. and by a grant from Russian Foundation for Basic Research No. 14-94-01446a to K.S.M.

References

  1. Kiseleva, L. L., Serebriiskaya, T. S., Horvath, I., Vigh, L., Lyukevich, A. A. and Los, D. A. (2000). Expression of the gene for the delta9 acyl-lipid desaturase in the thermophilic cyanobacterium. J Mol Microbiol Biotechnol 2(3): 331-338.
  2. Mironov, K. S., Sidorov, R. A., Kreslavski, V. D., Bedbenov, V. S., Tsydendambaev, V. D. and Los, D. A. (2014). Cold-induced gene expression and omega (3) fatty acid unsaturation is controlled by red light in Synechocystis. J Photochem Photobiol B 137: 84-88.
  3. Rippka, R. (1988). Isolation and purification of cyanobacteria. Methods Enzymol 167: 3-27.
  4. Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press.
  5. Sato, N. (1995). A family of cold-regulated RNA-binding protein genes in the cyanobacterium Anabaena variabilis M3. Nucleic Acids Res 23(12): 2161-2167.

简介

该方案描述了从蓝细菌集胞藻的细胞总RNA分离的程序。 PCC 6803。该方案也适用于细长聚球藻PCC 7942和PCC 6301,硫酸高热杆菌(Thermosynechococcus vulcanus)和其它单细胞和丝状物种的蓝细菌,其不具有含多糖的 外层。 对于后者,应该修改含有Trizol的协议。 RNA的产量取决于蓝细菌培养物的光密度,并且可以达到每1ml细胞培养物高达10-20μg的总RNA。 通过该方法分离的RNA可用于Northern印迹杂交,RT-qPCR,微阵列和下一代测序。

关键字:集胞藻, 蓝藻, RNA的分离, RNA的质量, 热酚

材料和试剂

  1. 在BG11培养基(Rippka,1988)中生长的蓝细菌培养物(50ml)(OD 750〜2)
  2. 95%乙醇用于分子生物学(保持在-20℃)
  3. 70%乙醇用于分子生物学(30ml RNA酶游离水和70ml 95%乙醇,保持在-20℃)
  4. 70%乙醇灭菌
  5. 苯酚BioUltra(用于分子生物学,TE饱和,〜73%)(Sigma-Aldrich,目录号:77607)(保持在4℃)
  6. 1M Tris-HCl(Life Technologies,目录号:15568-025)
  7. 0.5M EDTA-Na 2(pH 8.0)(Life Technologies,目录号:AM9260G)
  8. 无RNase高压灭菌水
  9. 氯仿(Sigma-Aldrich,目录号:C2432)(保持在4℃)
  10. 10M氯化锂溶液(Fluka,目录号:83268)(使用前在冰上冷却)
  11. 无RNA酶的DNAse
  12. 用于分子生物学的琼脂糖(Sigma-Aldrich,目录号:A9539)
  13. TAE电泳缓冲液(Sigma-Aldrich,目录号:T9650)
  14. RNA梯(Life Technologies,Invitrogen TM,目录号15620-016)
  15. TE缓冲区(参见配方)
  16. 50/100 TE缓冲液(见配方)
  17. 10 M LiCl水溶液(见配方)
  18. 单元修复解决方案(参见配方)

设备

  1. Saran包装(任何类型)
  2. 高压灭菌袋(Sigma-Aldrich,目录号:Z692212)
  3. 安全锁管(2.0ml)(Eppendorf,目录号:0030120.094)
  4. (Life Technologies,目录号:AM12501或Corning,Falcon)上的塑料锥形聚丙烯管(无菌,无核酸酶,无热原,高压灭菌)
  5. 高压灭菌器
  6. 带有铲斗转子的冷冻离心机适用于50ml锥形管(Eppendorf,型号:5804 R和带有50ml Falcon适配器的旋转斗转子A-4-44)
  7. 冷藏微量离心机预设为4°C(Eppendorf,型号:5415 R)
  8. 水浴预设为65°C
  9. Subzero冰箱:-20°C和-70°C
  10. 通风橱
  11. 冰浴
  12. 多管自动涡流(例如,微管混合器MT-400,Tomy Digital Biology)
  13. 紫外分光光度计(优选Nanodrop)
  14. 琼脂糖凝胶电泳装置(VWR Mini Gel II Complete Horizontal Electrophoresis System,目录号:95043-650)
    注意:在RNA分离程序之前,工作场所,移液器,手套等应该用70%乙醇清洗。 锥形管,Eppendorf管,移液管吸头应在使用前通过高压灭菌袋高压灭菌。

程序

  1. 探头固定
    1. 准备25毫升部分的细胞固定溶液在50ml塑料圆锥形 聚丙烯管。 保持在冰上。 倒入25毫升蓝细菌细胞 培养到25ml冰冷的细胞固定溶液中(图1.1)。 搅拌均匀   并在-20℃至1个月进行离心或储存样品。   对于50ml的蓝细菌培养物,应使用两个50ml管。 或者,100-200 ml离心瓶耐苯酚和 乙醇。
    2. 通过在4℃下以3,000xg离心5-10分钟收获固定的细胞(图1.2)。
    3. 丢弃细胞固定溶液留下0.5-1毫升的溶液重悬 颗粒。 通过涡旋或移液重悬沉淀。
    4. 将悬浮液转移到无菌的2毫升Eppendorf管中。 如果50ml管   用于细胞固定和收获,结合来自25的两个探针 ml管置于一个2ml Eppendorf管中。
    5. 离心机在3,000-5,000英寸x  g,4℃,3-5分钟,完全去除上清液(图1.3)。 离开颗粒细胞在冰上。
      注意:探头固定适用于 立即杀死细胞并避免RNA的可能降解。这是  当温度依赖性变化时特别重要 正在研究mRNA。


      图1. RNA提取步骤。 固定在细胞固定溶液中。 2.离心后固定的细胞。 3。 细胞沉淀在管底部。 4.细胞重悬于500μL  TE-50/100 5.加入1ml苯酚后的细胞悬浮液。 6.细胞 悬浮液。 7.在65℃水浴中温育。 8.四 离心后的相(从底部到顶部):蓝色沉淀(如果是) 绿色,热酚细胞裂解不完全;在这种情况下重新悬浮 并从步骤A4开始); 深绿色酚相; 蛋白 间隔(可能不可见); 粉红色TE-50/100相 酸。 小心地将上TE-50/100相转移到新管中。 使用 黄色提示。 10.不要使相间和苯酚相进入吸头。 11.加入300μl氯仿和300μl苯酚 TE-50/100-级分。 12.均匀白色乳液后 涡旋。 13.离心后的三相(从底部到顶部): 苯酚/氯仿(下相); 蛋白质间期; 粉色TE-50/100 相(上相)。 这个阶段应该转移到新的1.5 ml Eppendorf管。 14.重复步骤A9-13,直到相间消失。

  2. 热酚进行细胞裂解
    1. 通过涡旋或吸移在500μl无菌冰冷的50/100 TE缓冲液中重悬沉淀(图1.4)。
    2. 加入1ml苯酚,涡旋并在预热的水浴中孵育   65℃10分钟(图1.5-1.7)。 定期摇动探头(3-4次 孵育10分钟)以防止苯酚相分离。
    3. 在15-20℃下以最高速度(16,000xg)离心样品10分钟。 底部的沉淀应该是蓝色的,意味着细胞裂解   完成(图1.8)。
    4. 将上层水相转移到新的Eppendorf管中(图1.9-1.10)。

  3. 用苯酚/氯仿净化核酸
    1. 向探针中加入300μlTE饱和的苯酚和300μl氯仿(收集的水相)(图1.11)。
    2. 涡旋1-2分钟,并在16,000×g,4℃离心5分钟 (图1.12-1.13)。 对于多个样品应用多管自动化 涡流(例如,微管混合器MT-400)。
    3. 将上层水相转移到新管中。
    4. 重复此过程2-3次,直到中间相(发白, 变性的蛋白质层在苯酚相和水相之间 包含核酸的相)完全消失(图1.14)。

  4. 核酸沉淀
    1. 加入2-3倍体积的95%乙醇,预冷至-20-30℃至最终 水相(约1.5ml乙醇至〜0.5ml探针),充分搅拌 并在-20℃下孵育2-3小时至过夜。
    2. 在这个阶段,探针可以在-20℃下在乙醇下储存几个月。
    3. 离心机在最高速度,4℃下10分钟,仔细丢弃液体。
    4. 通过强烈涡旋洗涤核酸沉淀与1毫升70%冷乙醇。
    5. 以最高速度,4℃离心5-15分钟,通过抽吸完全除去乙醇。
    6. 重要的是在冰上用RNA进行所有操作

  5. 通过LiCl沉淀从RNA分离RNA
    1. 向沉淀中加入800μl50/100 TE缓冲液。 在冰上孵育15分钟。
    2. 涡旋探针3-4次,完全溶解沉淀。
    3. 加入200μl的10M LiCl溶液并通过涡旋混合。 离开探头冰上2小时。
    4. 以最高速度,4℃离心15-20分钟。
    5. 通过抽吸小心去除上清液。
    6. 通过加入1ml冰冷的70%乙醇洗涤RNA制剂 强烈涡流的丸。 以最高速度,4℃离心15分钟   min。
    7. 重复70%乙醇洗涤。
    8. 小心地删除所有 乙醇并在室温下在管架中空气干燥探针 覆盖由Saran套。 如果使用真空干燥器,请注意不要 干燥颗粒,因为它将变得不可能溶解它。   将RNA溶于50-100μl无RNA酶的水中

  6. RNA量和质量的评价
    1. 取1-2μl的RNA,并用Nanodrop分光光度计检查其浓度。
    2. LiCl不允许获得完全不含DNA的RNA,   但RNA的质量通常足以进行Nothern印迹分析。 如果 必要时,根据标准,用RNA酶额外纯化RNA样品   制造商说明。
    3. 通过琼脂糖凝胶检查RNA质量 电泳。电泳分离总蓝细菌RNA 可见为对应于23S,16S和5S RNA的3个主要条带。 线上的污迹指示在分离步骤期间RNA降解 (图2)。


      图2.从集胞藻中分离的总RNA样品。每泳道1μg的PCC 6803通过在1% 非变性琼脂糖凝胶上并用溴化乙锭染色。通常3  主要(或更多)条带对应于23S,16S和5S RNA 看到。泳道1-3代表高质量的分离的总RNA和泳道 4-6,涂抹的条带显示低质量的部分降解的RNA。对于 Northern印迹,RNA应在变性中分离 甲醛的1.2%琼脂糖凝胶上,转移 硝酸纤维素或尼龙膜(泳道7,8)。建议运行 两个额外的RNA样品泳道(泳道7)和RNA梯度(泳道8) 将被切断并用亚甲基蓝染色以确定尺寸 和印迹后RNA的质量。

笔记

LiCl溶液含有DNA,重要的是彻底清除上清液。 还可以通过2体积的乙醇或0.8体积的2-异丙醇(Lab Scan,C19C11X)沉淀LiCl溶液中的DNA。

食谱

  1. TE缓冲区
    10mM Tris-HCl(pH7.5-8.0) 1mM EDTA-Na 2
  2. 50/100 TE缓冲区
    50mM Tris-HCl(pH 7.5-8.0)
    100 mM EDTA
    制备后,该缓冲液应在121℃,1.5大气压,15分钟的高压灭菌
  3. 10 M LiCl水溶液中 注意:应将其高压灭菌,在无菌条件下分装,并保存无菌。
  4. 单元格修复解决方案
    0.5%苯酚在95%乙醇中的溶液 在高压灭菌的50ml试管中的25ml等分试样应贮存在-20℃下

致谢

这项工作是由俄罗斯科学基金会14-24-00020授予D.A.L.以及俄罗斯基础研究基金会第14-94-01446a号给K.S.M的资助。

参考文献

  1. Kiseleva,L.L.,Serebriiskaya,T.S.,Horvath,I.,Vigh,L.,Lyukevich,A.A。和Los,D.A。(2000)。 在嗜热蓝细菌中表达delta9酰基 - 脂质去饱和酶的基因 。 J Mol Microbiol Biotechnol 2(3):331-338。
  2. Mironov,K. S.,Sidorov,R. A.,Kreslavski,V. D.,Bedbenov,V. S.,Tsydendambaev,V. D. and Los,D.A。(2014)。 冷诱导的基因表达和ω(3)脂肪酸不饱和度由红光控制 Synechocystis 。 J Photochem Photobiol B 137:84-88。
  3. Rippka,R。(1988)。 蓝细菌的分离和纯化方法Enzymol 167: 3-27。
  4. Sambrook,J.,Fritsch,E.F.and Maniatis,T。(1989)。 分子克隆:实验室手册。冷泉港实验室出版社。
  5. Sato,N。(1995)。 蓝藻中的冷调节的RNA结合蛋白基因家族 Anabaena variabilis em> M3。 Nucleic Acids Res 23(12):2161-2167。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Mironov, K. S. and Los, D. A. (2015). RNA Isolation from Synechocystis. Bio-protocol 5(6): e1428. DOI: 10.21769/BioProtoc.1428.
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Tarkeshwar Singh
IISER KOLKATA
Hi!

I am using Trizol method for extracting cyanobacterial RNA, their I am facing problem in phase separation step. What should be the region for that?
5/10/2016 3:35:00 AM Reply
Kirill Mironov
Russian Academy of Sciences

Hi, Tarkeshwar, I have never tried to use Trizol for RNA isolation from cyanobacteria

5/10/2016 3:45:36 AM