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Yeast DNA Replication 2D Gel Protocol
适于二维琼脂糖凝胶电泳检测复制起始的酵母DNA提取   

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Abstract

Two-dimensional agarose gel electrophoresis (2D gel) analysis is used extensively as a method to detect origins of replication. Here, I present a simplified method for the isolation of yeast genomic DNA for 2D gel analysis from a small number of yeast cells. This DNA isolation method is simpler and less time consuming than the traditional method that involves CsCl density gradient centrifugation. This method could be modified for 2D gel analysis in other organisms as well.

Materials and Reagents

  1. Qiagen genomic-tip 20/G ( QIAGEN, catalog number: 10223 , if more DNA needed, 100/G could be used to isolate DNA)
  2. Qiagen Buffer G2 ( QIAGEN, catalog number: 1014636 )
  3. Qiagen buffer QBT (QIAGEN, catalog number: 19054 )
  4. Qiagen buffer QC (QIAGEN, catalog number: 19055 )
  5. Qiagen buffer QF ( QIAGEN, catalog number: 19056 )
    Tips: Buffer QBT, QC and QF could be made by yourself.
  6. RNase A (10 mg/ml) ( QIAGEN, catalog number: 19103 )
  7. Proteinase K (20 mg/ml) (QIAGEN, catalog number: 19131 )
    Tips: RNase A and Proteinase K could be ordered from any other company, they work fine too.
  8. Sodium azide
  9. Isopropanol
  10. Tris-HCl
  11. EDTA
  12. Agarose
  13. KoAc
  14. Ethidium bromide
  15. Glycerol
  16. MgCl2
  17. Spermine
  18. Spermidine
  19. KOH
  20. GuHCl
  21. MOPS free acid
  22. Tween-20
  23. Triton X-100
  24. NaCl
  25. KB ladder
  26. 70% ethanol
  27. TE (see Recipes)
  28. Nuclear isolation buffer (NIB buffer) (see Recipes)
  29. Sodium azide solution (see Recipes)
  30. Qiagen Buffer QBT (see Recipes)
  31. Qiagen Buffer QC (see Recipes)
  32. Qiagen Buffer QF (see Recipes)

Equipment

  1. Acid-washed glass beads (425-600 μm diameter) (Sigma-Aldrich, catalog number: G8772-500G )
  2. BioRad sub-cell GT gel box or other Maxi horizontal electrophoresis units (big agarose gels are needed for both dimentions)
  3. 4 °C cold room
  4. Micro centrifuges
  5. Vortex mixer (VORTEX-GENIE)
  6. Microwave oven
  7. Hand-held UV lamp
  8. UV light box
  9. 2D gel apparatus tray (4 samples per apparatus -- Fischer self-circulating gel box)
  10. 50 ml polypropylene centrifuge tubes
  11. 2-ml polypropylene tube
  12. Pasteur pipette
  13. 12 x 75 mm polypropylene culture tube

Procedure

  1. Day 1: Solutions and preparation, as described in Materials and Reagents, and Recipes sections.

  2. Days 1-2: Yeast culture growth
    1. Pick a single colony or part of a single colony. Inoculate 5 ml cultures in the appropriate medium at the appropriate temperature.
    2. Enlarge the cell cultures after 1 overnight to OD600~0.1-0.2 in 50 ml medium.
    3. Grow until they reach log phase. Follow growth progress by monitoring OD600 over time: Harvest the cells when OD600 is between 0.6-1.0.
    4. Treat 50 ml cultures with 0.5 ml of 0.1 g/ml sodium azide (4.25 g NaAzide in 42.5 ml sterile H2O). Swirl well. Place on ice for 5-10 min.
    5. Harvest cells by centrifugation in 50 ml polypropylene centrifuge tubes, wash once with 20-30 ml of ice-cold distilled water and resuspend pellet in 0.8 ml ice-cold nuclear isolation buffer (NIB).
    6. Place cultures at -80 °C if doing the prep another day OR proceed to step 2 of the Genomic DNA purification protocol (below).

  3. Day 3: Genomic DNA Purification
    1. Thaw cultures from -80 °C freezer (frozen in NIB in the 2-ml polypropylene tube).
    2. An equal volume (800 μl) of acid-washed glass beads was added, and the cell suspension was vigorously shaken with a Vortex-Genie in the cold room at maximum speed for 1 min, and stood on ice for 1 min. This routine was repeated 12-20 times, until >90% of cells were broken (lysis monitored by phase contrast microscopy).
    3. Broken cells were removed with a Pasteur pipette to a 12 x 75 mm polypropylene culture tube, and the glass beads were rinsed twice with 0.8 ml ice-cold NIB and transferred to the same tube (it does not matter if some glass beads get transferred).
    4. Broken cells were separated into 2 of 1.5 ml eppendorf tubes and pelleted at 6,500 x g at 4 °C for 10 min and the pellet in each tube was dissolved in 1 ml Qiagen buffer G2 containing 20 μl RNase A (10 mg/ml).
    5. It is important to generate a homogeneous lysate by gently inverting the tube ~20 times (do not vortex).
    6. The lysate was incubated at 37 °C for 30 min, then 25 μl proteinase K (20 mg/ml) was added, and the lysate was incubated for 60 min at 42 °C.
    7. After incubation, the lysate was centrifuged at  6,500 x g at 4 °C for 10 min.
    8. A Qiagen column (20/G) was equilibrated with 2 ml Buffer QBT (allow the QIAGEN Genomic-tip empty by gravity flow).
    9. The supernatant from step 7 should be clear. Carefully transfer the supernatant to a 12 x 75 mm polypropylene culture tube. Pool the supernatants from the two tubes together.
      Note: Take a 50 μl aliquot of the supernatant and save it for an analytic gel (aliquot 1).
    10. Add an equal volume of Qiagen Buffer QBT to the supernatant and mix it by gently inverting the tube or Vortex it for 10 sec at full speed. Then load the sample to the equilibrated column and allowed to pass through by gravity flow.
      Note: Take a 100 μl aliquot of the flow through and save it for an analytic gel (aliquot 2).
    11. Wash the Column with 3 x 1 ml of Buffer QC.
      Note: Take a 300 μl aliquot of the flow through and save it for an analytic gel (aliquot 3).
    12. Elute the genomic DNA with 2 x 1 ml of Buffer QF. Use of Buffer QF pre-warmed to 50 °C will increase yields.
      Note: Take a 50 μl aliquot of the flow through and save it for an analytic gel (aliquot 4).
    13. Precipitate the DNA by adding 1.4 ml (0.7 volumes) room temperature isoproponal to the eluted DNA.
    14. The solution was well mixed and divided into two or three 1.5 ml Eppendorf tubes.
    15. Precipitate DNA by centrifugation in a microfuge at full speed for 15 min at 4 °C.
    16. The supernatants were discarded, 500 μl 70% ethanol (RT) was added to each tube.
    17. DNA was again pelleted for 2 min in a microfuge at full speed and most of the 70% ethanol was discarded. The tube was again centrifuged at full speed for 3-5 sec and all traces of 70% ethanol were removed.
    18. Air-dry for 5-10 min and resuspend DNA in 40 μl TE at RT for 1-2 h or at 4 °C overnight. If the DNA pellet is allowed to dry at this step, resuspension will be very difficult.
    19. At least three times during this time gently flick the tubes to promote resuspension of the genomic DNA. It is ok to SLOWLY pipette up and down using large orifice tips.

  4. Day 4: Resuspension and pooling of genomic DNA
    1. After 24-30 h pool all the tubes for each sample (large orifice tips) and store at 4 °C for an additional 12+ h. Mix by slow pipetting up and down using large orifice tips.
    2. Do the analytic gel to determine yield, purity and length of DNA.
      Precipitate each of aliquots with 0.7 volumes of isopropanal.
      Rinse the pellets with 70% ethanol, drain well and resuspend in 20 μl of TE.
      Use all 20 μl for analysis on a 0.5% agarose gel. Run the gel until the blue dye is near the bottom, and stain it in EtBr solution.

  5. Day 6: Digestion of genomic DNA & starting the first dimension.
    1. Set up to digest ~10 μg of DNA per sample in a 600 μl reaction volume:
      DNA + ddH2O should be equal a total volume of 150 μl, so add the appropriate volume of DNA to each tube to make 10 μg and bring the volume up to 150 μl.
      Enzyme mix should be in a volume of 450 μl per sample. DNA is typically digested with 300-400 units of enzyme for 5-6 h at 37 °C.
    2. Mix DNA with enzyme mixes by gently pipetting up and down using large orifice tips.
      Note: Do not vortex your samples.
    3. Digest DNA 5 h at 37 °C. Every hour gently mix DNA in tube by lightly flicking the tube and the bumping down and placing back at 37 °C.
    4. While DNA is digesting, make your 0.35% (w/v) agarose gel mix: 400 ml 1x TBE + 1.4 g Agarose in a 1 L glass bottle. Heat in the microwave until all agarose has gone into solution. Check for complete resuspension by swirling the bottle and looking at the bottom for agarose that is not fully in solution. Store at 55 °C for about 30-40 min to cool to a good pouring temperature. Pour the gel in the cold room. The gel tray should be 15 cm x 25 cm and should be leveled within the BioRad sub-cell GT gel box.1 gel allows for up to 8 samples. Once solidified, move gel to room temp. Set up for first dimension by adding the 1.6-1.8 L of 1x TBE needed to fill the gel box and then remove the comb.
    5. Confirm complete digestion by running an agarose gel of your digested DNA alongside undigested genomic DNA on a small 0.5% gel. Room for photo of gel(s) below.
    6. Precipitate digested DNA:
      1. Add 60 μl of 3 M KoAc (pH 5.5).
      2. Add 700 μl of 100% isopropanol.
      3. Mix gently by slowly inverting the tube several times.
    7. Spin down your precipitated DNA for 20 min at 14,000 rpm and 4 °C. Pour off the supernatent and wash the pellet 2x with 1 ml of 70% EtOH (5 min spins each time as above). Remove as much of the second wash as possible with a p200. Dry on the bench-top ~10 min. Resuspend with 16 μl of 1x TE (pH 8.0). Do not vortex. Resuspend by flicking the tube very gently and leaving on ice or at RT for ~1 h. After 1 h add 7 μl of 5x dyes, mix gently and bump down.
    8. Load KB ladder in the first lane. Skip two lanes and then load 20 μl of the DNA samples in every other lane.
    9. When done loading, start the gel. Run the gel 42-48 h at 22 V at RT.
    10. Set up for the next dimension by pre-chilling 2 L of 1x TBE for every 4 samples to be run. Place 4 L of buffer in the cold room. N.B. Add 60 μl of 10 mg/ml ethidium bromide to every 2 L of pre-chilled 1x TBE (0.3 µg/ml ethidium bromide final concentration) either now or right before using.

  6. Day 8: Starting the second dimension
    1. Make your 0.95% (w/v) agarose second dimension gel mixes: 500 ml of 1X TBE + 4.75 g of agarose + 15 μl of 10 mg/ml ethidium bromide (0.3 µg/ml final concentration). Mix in a 1 L glass bottle. Microwave on high until all agarose has gone into solution. Check by swirling the bottle and looking at the bottom. There should be no denser agarose visible. Store at 55 °C until ready to pour the second dimension.
    2. At the appropriate time stop the first dimension. Carefully soak the gel in a glass baking dish with 750 ml of 1x TBE with 0.3 µg/ml ethidium bromide to stain the DNA. This should take 30-40 min. The gel is very fragile, so be extra careful.
    3. Excise the KB ladder lane and photograph on the eagle-eye. Photograph a ruler alongside the ladder and overlay the two. Determine which 9-10 cm slice of the gel to run in the second dimension and excise by cutting between the lanes and with the right length. It is easiest to cut out the 9 cm slices first and then to cut between the lanes while on the UV light box. Try to cut with the UV setting on preparative (long wave) to avoid nicking. You can show the KB ladder and ruler in the space provided below:
    4. Transfer the slices to the 2D gel apparatus tray (4 samples per apparatus -- Fischer self-circulating gel box). Place the DNA so that the higher molecular weights are to the left. Set up the apparatus in the cold room and level it.
    5. Seal the edges of the gel and seal the slices in place with agarose. Pour the second dimension gel and make sure the gel slices do not move. Allow to solidify approximately 45 min to 1 h. Remove the gel dams and add the pre-chilled 2 L of 1x TBE and run the gel 18-30 h (depending on fragment size) at 130 V.
    6. You can monitor the progress in the second dimension with a hand-held UV lamp. Run the gel so that the smallest fragments on the arc of linear molecules are just reaching the bottom of the gel (lower right hand corner of the gel).

  7. Day 9: Stop second dimension and southern transfer
    1. Stop the gels. Cut the four gels apart in the middle, leaving you with two sets of two gels. Place in glass baking dishes and be sure to label the glass baking dishes with what is on each gel. Photograph the gels on the eagle eye (or UV light box).
    2. Paste gel photo(s) here.
    3. Go to the normal transfer process and southern blot steps.

Recipes

  1. Nuclear isolation buffer (NIB buffer)
    68 ml of 100% glycerol (17% v/v)
    4.2 g of MOPS free acid (50 mM)
    5.88 g of KoAc (150 mM)
    0.8 ml of 1 M MgCl2 (2 mM)
    20.8 mg of Spermine (150 µM)
    200 μl of 1 M Spermidine (500 µM)
    Add ddH2O to 375 ml, pH to 7.2 with KOH, add ddH2O to 400 ml.
    Note: Do not autoclave. Store at 4 °C.
  2. Sodium Azide (0.1 g/ml, 5 ml/500 ml culture)
    42.5 ml ddH2O + 4.25 g Na-Azide
    Vortex, store at 4 °C.
  3. Qiagen Buffer G2 ( QIAGEN) (Bought) (pH 8.0)
    800 mM GuHCl
    30 mM EDTA
    30 Mm Tris-HCl
    5% Tween-20
    0.5% Triton X-100
  4. Qiagen Buffer QBT (pH 7.0)
    750 mM NaCl
    50 mM MOPS
    15% ethanol
    0.15% Triton X-100
  5. Qiagen Buffer QC (pH 7.0)
    1.0 M NaCl
    50 mM MOPS
    15% ethanol
  6. Qiagen Buffer QF (pH 8.5)
    1.25 M NaCl
    50 mM Tris-HCl
    15% ethanol
  7. TE (pH 8.0)
    10 mM Tris-HCl
    1 mM EDTA

Acknowledgments

This protocol was adapted from Zou and Bi (2008) and Wu and Gilbert (1995).

References

  1. Wu, J. R. and Gilbert, D. M. (1995). Rapid DNA preparation for 2D gel analysis of replication intermediates. Nucleic Acids Res 23(19): 3997-3998.
  2. Zou, Y. and Bi, X. (2008). Positive roles of SAS2 in DNA replication and transcriptional silencing in yeast. Nucleic Acids Res 36(16): 5189-5200.

简介

二维琼脂糖凝胶电泳(2D凝胶)分析广泛用作检测复制起点的方法。 在这里,我提出了一个简单的方法分离酵母基因组DNA的2D凝胶分析从少量的酵母细胞。 这种DNA分离方法比涉及CsCl密度梯度离心的传统方法更简单和更省时。 这种方法可以修改为其他生物体的2D凝胶分析。

材料和试剂

  1. Qiagen基因组提示20/G( QIAGEN,目录号:10223 ,如果需要更多的DNA,100/G可用于分离DNA)
  2. Qiagen缓冲液G2( QIAGEN,目录号:1014636
  3. Qiagen缓冲液QBT( QIAGEN,目录号:19054
  4. Qiagen缓冲液QC( QIAGEN,目录号:19055
  5. Qiagen缓冲液QF( QIAGEN,目录号:19056
    提示:缓冲QBT,QC和QF可以自己完成。
  6. 核糖核酸酶A(10 mg/ml)( QIAGEN,目录号:19103
  7. 蛋白酶K(20 mg/ml)( QIAGEN,目录号:19131 )
    提示:RNase A和Proteinase K可以从任何其他公司订购,他们也很好。
  8. 叠氮化钠
  9. 异丙醇
  10. Tris-HCl
  11. EDTA
  12. 琼脂糖
  13. KoAc
  14. 溴化乙锭
  15. 甘油
  16. MgCl 2
  17. 精胺
  18. 亚精胺
  19. KOH
  20. GuHCl
  21. MOPS游离酸
  22. 吐温-20
  23. Triton X-100
  24. NaCl
  25. KB梯形图
  26. 70%乙醇
  27. TE(参见配方)
  28. 核分离缓冲液(NIB缓冲液)(参见配方)
  29. 叠氮化钠溶液(参见配方)
  30. Qiagen Buffer QBT(参见配方)
  31. Qiagen Buffer QC(参见配方)
  32. Qiagen Buffer QF(参见配方)

设备

  1. 酸洗玻璃珠(直径425-600μm)(Sigma-Aldrich,目录号:G8772-500G)
  2. BioRad亚细胞GT凝胶盒或其他Maxi水平电泳装置(两种尺寸都需要大的琼脂糖凝胶)
  3. 4°C冷室
  4. 微离心机
  5. 涡流混合器(VORTEX-GENIE)
  6. 微波炉
  7. 手持紫外灯
  8. 紫外线灯箱
  9. 2D凝胶装置托盘(每个装置4个样品 - Fischer自循环凝胶盒)
  10. 50ml聚丙烯离心管
  11. 2 ml聚丙烯管
  12. 巴斯德移液器
  13. 12×75mm聚丙烯培养管

程序

  1. 第1天:解决方案和准备,如材料和试剂和食谱部分所述。

  2. 第1-2天:酵母培养物生长
    1. 选择单个菌落或单个菌落的一部分。 在适当的温度下在适当的培养基中接种5ml培养物
    2. 在1小时后将细胞培养物放大至在50ml培养基中的OD 600 = 0.1-0.2。
    3. 生长直到它们达到对数期。 通过随时间监测OD 600随着生长进展:当OD 600为0.6-1.0时收获细胞。
    4. 用0.5ml 0.1g/ml叠氮化钠(4.25g NaAzide在42.5ml无菌H 2 O中)处理50ml培养物。 旋转很好。 置于冰上5-10分钟。
    5. 通过在50ml聚丙烯离心管中离心收获细胞,用20-30ml冰冷的蒸馏水洗涤一次,并将沉淀重悬于0.8ml冰冷的核分离缓冲液(NIB)中。
    6. 将培养物置于-80°C,如果再次做准备,或进行基因组DNA纯化方案的步骤2(下面)。

  3. 第3天:基因组DNA纯化
    1. 解冻培养物从-80℃冰箱(冷冻在NIB在2-ml聚丙烯管中)
    2. 加入等体积(800μl)的酸洗玻璃珠,并将细胞悬浮液用Vortex-Genie在冷室中以最大速度剧烈摇动1分钟,并在冰上放置1分钟。 该程序重复12-20次,直到> 90%的细胞破裂(通过相差显微镜监测裂解)。
    3. 使用巴斯德移液管将破碎的细胞移出到12×75mm聚丙烯培养管中,并且用0.8ml冰冷的NIB冲洗玻璃珠两次并转移到同一管中(如果一些玻璃珠被转移,则是无关紧要的) 。
    4. 将破碎的细胞分离成2个1.5ml的eppendorf管,并在6,500×g下在4℃沉淀10分钟,并将每个管中的沉淀物溶解于1ml含有20μlRNase A( 10 mg/ml)
    5. 重要的是通过轻轻颠倒试管约20次(不要涡旋)产生均匀的裂解物
    6. 将裂解物在37℃下孵育30分钟,然后加入25μl蛋白酶K(20mg/ml),裂解物在42℃下孵育60分钟。
    7. 孵育后,将裂解物离心, 6,500×g 在4℃下10分钟
    8. 用2ml缓冲液QBT(允许QIAGEN基因组 - 末端通过重力流动为空)使Qiagen柱(20/G)平衡。
    9. 来自步骤7的上清液应该是澄清的。小心地将上清液转移到12×75mm聚丙烯培养管中。将两个管中的上清液汇集在一起​​。
      注意:取上清液的50μl等分试样,并保存为分析凝胶(等分试样1)。
    10. 加入等体积的Qiagen缓冲液QBT到上清液中,轻轻倒转管或涡旋混合10秒,全速。然后将样品装载到平衡柱上,并通过重力流通过 注意:取100μl等分试样,并保存为分析凝胶(等分试样2)。
    11. 用3×1ml缓冲液QC洗涤柱子。
      注意:取300μl等分试样,并保存为分析凝胶(等分试样3)。
    12. 用2×1ml的Buffer QF洗脱基因组DNA。使用预热至50°C的缓冲液QF将提高产率 注意:取50μl等分试样,并保存为分析凝胶(等分试样4)。
    13. 通过向洗脱的DNA中加入1.4ml(0.7体积)室温异丙醇沉淀DNA
    14. 将溶液充分混合并分成两个或三个1.5ml Eppendorf管
    15. 通过在微量离心机中在4℃下全速离心15分钟沉淀DNA
    16. 弃去上清液,向每个管中加入500μl70%乙醇(RT)
    17. 将DNA再次在微量离心机中以全速度沉淀2分钟,并丢弃大部分的70%乙醇。 将管再次全速离心3-5秒,除去所有痕量的70%乙醇。
    18. 空气干燥5-10分钟,并将DNA在40μlTE中在室温下重悬1-2小时或在4℃过夜。 如果DNA沉淀在此步骤中干燥,则重悬将是非常困难的
    19. 在此期间至少三次轻轻地轻拂管以促进基因组DNA的重悬。 可以使用大口径的吸头向上和向下缓慢移液。

  4. 第4天:重悬和合并基因组DNA
    1. 24-30小时后,将每个样品的所有管(大孔尖端)集中并在4℃下储存另外12小时。 使用大孔口尖端通过缓慢移液上下混合。
    2. 做分析凝胶以确定DNA的产量,纯度和长度 用0.7体积的异丙醇沉淀每个等分试样 用70%乙醇冲洗沉淀,排空,并重悬于20μlTE 使用所有20μl在0.5%琼脂糖凝胶上分析。 运行凝胶,直到蓝色染料靠近底部,并在EtBr溶液中染色。

  5. 第6天:基因组DNA消化开始第一维。
    1. 设置为在600μl反应体积中消化〜10μgDNA /样品:
      DNA + ddH 2 O的总体积应等于150μl,因此向每个试管中加入适当体积的DNA以制备10μg,并将体积增至150μl。
      酶混合物应该在每个样品450μl的体积。 DNA通常在37℃下用300-400单位的酶消化5-6小时
    2. 通过使用大孔尖端轻轻吹打上下混合DNA与酶混合物 注意:不要旋转样品。
    3. 消化DNA在37℃下5小时。每小时轻轻地轻轻甩管,然后放回到37°C,轻轻地将DNA轻轻地混合在管中。
    4. 当DNA消化时,在1L玻璃瓶中制备0.35%(w/v)琼脂糖凝胶混合物:400ml 1x TBE + 1.4g琼脂糖。在微波中加热,直到所有琼脂糖进入溶液。检查是否完全重悬,通过旋转瓶子,并看看底部的琼脂糖,不完全在溶液中。在55℃下储存约30-40分钟以冷却至良好的浇注温度。将凝胶倒入冷室。凝胶托盘应为15 cm×25cm,并且应该在BioRad子细胞GT凝胶盒1中平整。凝胶允许最多8个样品。 一旦固化,将凝胶移至室温。 通过添加填充凝胶盒所需的1.6-1.8升1x TBE,然后取出梳子,设置第一维。
    5. 通过在小的0.5%凝胶上运行消化的DNA的琼脂糖凝胶以及未消化的基因组DNA来确认完全消化。 以下凝胶照片室。
    6. 沉淀消化的DNA:
      1. 加入60μl3 M KoAc(pH 5.5)
      2. 加入700μl100%异丙醇。
      3. 慢慢倒转管子几次,轻轻混匀。
    7. 在14,000 rpm和4℃下,将沉淀的DNA旋转20分钟。倒出上清液并用1ml 70%EtOH(每次如上所述5分钟旋转)洗涤沉淀2次。使用p200尽可能多地去除第二次洗涤。在台面上干燥〜10分钟。用16μl1x TE(pH 8.0)重悬。不要涡旋。通过轻轻地轻轻地轻轻地重悬,并在冰上或在室温下放置约1小时。 1小时后加入7μl5x染料,轻轻混匀并向下冲洗
    8. 在第一个通道中装入KB梯形图。跳过两个泳道,然后加载20μl的DNA样品在每个其他泳道
    9. 完成加载后,启动凝胶。在室温下在22V下运行凝胶42-48小时
    10. 通过每运行4个样品预冷2升1x TBE,设置下一个维度。将4 L缓冲液放在冷室中。 N.B.在使用前或现在或之后,向每2L预冷冻的1×TBE(0.3μg/ml溴化乙锭终浓度)中加入60μl的10mg/ml溴化乙锭。

  6. 第8天:开始第二个维度
    1. 制备0.95%(w/v)琼脂糖第二维凝胶混合物:500ml的1×TBE + 4.75g的琼脂糖+15μl的10mg/ml溴化乙锭(0.3μg/ml终浓度)。在1L玻璃瓶中混合。微波高,直到所有的琼脂糖已经进入溶液。通过旋转瓶子,看看底部。应该没有可见的更稠密的琼脂糖。储存在55°C,直到准备好倾倒第二个维度。
    2. 在适当的时间停止第一维。小心地将凝胶浸泡在玻璃烤盘中,用750ml含有0.3μg/ml 1×TBE的 溴化乙锭染色DNA。这应该需要30-40分钟。凝胶非常脆弱,所以要特别小心。
    3. 消除KB梯子线和鹰眼上的照片。沿着梯子拍摄一个尺子,然后重叠两个。确定哪个9-10厘米的凝胶切片在第二维中运行,并通过在车道之间切割并以正确的长度切除。最容易的是首先切出9厘米的切片,然后在紫外线灯箱上切割通道。尝试用制备(长波)上的UV设置切割,以避免切口。您可以在以下空格中显示KB梯子和标尺:
    4. 将切片转移到2D凝胶装置托盘(每个装置4个样品 - 费歇尔自循环凝胶盒)。放置DNA,使较高的分子量在左边。在寒冷的房间里设置设备和水平。
    5. 密封凝胶的边缘,并用琼脂糖将切片密封在适当位置。 倒入第二维凝胶并确保凝胶切片不移动。 允许固化约45分钟至1小时。 取出凝胶坝,加入预冷的2 L 1x TBE,并在130 V下运行凝胶18-30小时(取决于片段大小)。
    6. 您可以使用手持紫外灯监视第二维的进度。 运行凝胶,使线性分子弧上的最小碎片正好到达凝胶底部(凝胶右下角)。

  7. 第9天:停止第二维和南方传送
    1. 停止凝胶。 在中间切割四个凝胶分开,留下两组两个凝胶。 放在玻璃烤盘,并确保标签玻璃烤盘与什么是每个凝胶。 拍摄鹰眼上的凝胶(或紫外线灯箱)。
    2. 在这里粘贴凝胶照片。
    3. 转到正常的转移过程和Southern印迹步骤

食谱

  1. 核分离缓冲液(NIB缓冲液)
    68ml的100%甘油(17%v/v) 4.2g MOPS游离酸(50mM) 5.88g KoAc(150mM) 0.8ml的1M MgCl 2(2mM)
    20.8mg精胺(150μM)
    200μl的1M亚精胺(500μM)
    将ddH 2 O加至375ml,用KOH将pH调至7.2,将ddH 2 O加至400ml。
    注意:不要高压灭菌。 存储于4°C。
  2. 叠氮化钠(0.1g/ml,5ml/500ml培养物) 42.5ml ddH 2 O + 4.25g叠氮化钠 涡旋,储存在4°C
  3. Qiagen缓冲液G2(QIAGEN)(购买)(pH8.0) 800 mM GuHCl 30 mM EDTA
    30 Mm Tris-HCl
    5%Tween-20
    0.5%Triton X-100
  4. Qiagen缓冲液QBT(pH 7.0)
    750mM NaCl 50 mM MOPS
    15%乙醇 0.15%Triton X-100
  5. Qiagen Buffer QC(pH 7.0)
    1.0 M NaCl
    50 mM MOPS
    15%乙醇
  6. Qiagen Buffer QF(pH 8.5)
    1.25 M NaCl
    50mM Tris-HCl
    15%乙醇
  7. TE(pH 8.0)
    10mM Tris-HCl
    1mM EDTA

致谢

该协议改编自Zou和Bi(2008)和Wu和Gilbert(1995)。

参考文献

  1. Wu,J.R。和Gilbert,D.M。(1995)。 快速DNA制备用于复制中间体的2D凝胶分析。核酸研究 23(19):3997-3998。
  2. Zou,Y。和Bi,X。(2008)。 SAS2在DNA复制和转录沉默在酵母中的积极作用核 Acids Res 36(16):5189-5200。
  • English
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zou, Y. (2012). Yeast DNA Replication 2D Gel Protocol. Bio-protocol 2(13): e213. DOI: 10.21769/BioProtoc.213.
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