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Generation of Non-typeable Haemophilus influenzae Directed Gene Deletion Mutants
非分型流感嗜血杆菌定向基因缺失突变体的构建   

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参见作者原研究论文

本实验方案简略版
Journal of Bacteriology
Jun 2013

Abstract

Directed deletion mutants in non-typeable Haemophilus influenzae can be made by allelic exchange of the target gene with an artificial DNA construct in which an antibiotic resistance cassette is placed between two ~1,000 bp DNA sequences that are identical to the 5' and 3' flanking regions of the target gene. The artificial DNA construct that is required for this mutagenesis is synthesized by the so-called Megaprimer PCR method (Figure 1).

Keywords: Haemophilus influenzae (流感嗜血杆菌), Gene deletion (基因缺失), Mutants (突变体), Mutagenesis (诱变), Molecular microbiology (分子微生物学)



Figure 1. Schematic representation of the Megaprimer PCR method. Step A1. The flanking regions (~1,000 bp) of the target gene are amplified by PCR (Primer pair L1 + L2 and R1 + R2). The L2 and R2 primers have extensions with homology to the antibiotic resistance cassette. Step A2. The antibiotic resistance cassette is amplified by PCR (primer pair L + R). Step B. The three PCR products of the first PCR reactions are combined. The flanking regions anneal to the antibiotic resistance cassette and one large PCR product is formed.

Materials and Reagents

  1. Amplitaq DNA polymerase including 25 mM MgCl2 and 10x reaction buffer (Applied Biosystems®, catalog number: N8080171 )
  2. PWO DNA polymerase including 25 mM MgSO4 and 10x reaction buffer (Roche Diagnostics, catalog number: 11644955001 )
  3. 10 mM dNTP mix
  4. Sterile Milli-Q water
  5. QIAquick PCR purification kit (QIAGEN, catalog number: 28104 )
  6. Agarose
  7. Ethidium bromide
  8. 0.5x TBE
  9. 100 bp perfect DNA ladder (Merck Millipore, catalog number: 70539 )
  10. 1 kb perfect DNA ladder (Merck Millipore, catalog number: 70537 )
  11. 1x PBS
  12. Bacto-agar (BD Biosciences, catalog number: 212030 )
  13. Bacto-Brain Heart Infusion medium (BD Biosciences, catalog number: 237500 )
  14. Glycerol
  15. M-IV competent non-typeable Haemophilus influenzae (Herriott et al., 1970), for protocol see below.

Equipment

  1. T100 thermal cycler (Bio-Rad Laboratories)
  2. Nanodrop spectrophotometer (Thermo Fisher Scientific, Nanodrop, model: ND1000 )
  3. Centrifuge (Eppendorf, model: 5810 )
  4. Microcentrifuge (Eppendorf, model: 5417R )
  5. Shaker (New Brunswick Scientific, model: Innova 4000 )
  6. 37 °C, 5% CO2 incubator (BINDER GmbH, model: CB 150 )
  7. DNA gel electrophoresis equipment

Procedure

  1. PCR amplify the antibiotic resistance cassette and the flanking regions of the target gene.
    Please see Figure 1 about primer pairs, L1/L2, R1/R2 and L/R used in this PCR.
    Reaction mix:
    5 μl 10x PWO polymerase buffer (-MgSO4)
    3 μl 25 mM MgSO4*
    5 μl 2.5 mM dNTPs
    5 μl 4 pmol/μl primer L1, R1, or L, respectively
    5 μl 4 pmol/μl primer L2, R2, or R, respectively
    1 μl 5 ng/μl template DNA*#
    0.5 μl PWO DNA polymerase
    25.5 μl dH2O
    Vtot = 50 μl
    PCR cycle:
    93 °C - 4 min
    93 °C 30 sec   |
    55 °C 1 min    | 35x
    68 °C 1.5 min |
    68 °C 5 min
    12 °C ∞
    * Optimize if necessary. Increased MgSO4 or DNA template concentrations might increase yield, but might also decrease specificity.
    # Template for the flanking regions is chromosomal DNA. Template for the antibiotic resistance cassette can be plasmids or chromosomal DNA of a resistant strain.
  2. Purify PCR products with QIAquick PCR Purification Kit.
  3. Measure DNA concentration with the Nanodrop. Concentration of >20 ng/μl is enough for the Megaprimer PCR (step 6).
  4. Load 2 μl PCR product mixed with DNA loading buffer on a 1.5% agarose gel with a 100 bp DNA marker.
  5. Check the gel for purity (should be a single PCR product) and size of the product (size depends on the primer design, usually ~1,000 bp). When the desired product is not amplified, repeat the PCR in step 1 with modified conditions (e.g. decreased Tm, increased MgSO4 or addition of DMSO).
  6. Perform a MegaPrimer PCR to unite the left and right flanking region of the target gene with the antibiotic resistance cassette.
    Reaction mix:
    5 μl 10x PWO polymerase buffer (+20 mM MgSO4)
    5 μl 2.5 mM dNTPs
    ~200 ng PCR product L1+ L2 (left flanking region)
    ~200 ng PCR product R1+ R2 (right flanking region
    ~400 ng PCR product R + L (antibiotic resistance cassette)
    0.5 μl PWO DNA polymerase
    Vtot = 50 μl with dH2O
    PCR cycle:
    93 °C 4 min
    93 °C 30 sec |
    55 °C 1 min  | 30x
    68 °C 3 min  |
    68 °C 7 min
    12 °C ∞
  7. Load 2 μl PCR product mixed with DNA loading buffer on a 0.7% agarose gel with a 1 kb DNA marker.
  8. Check the gel for purity and size of the PCR product. PCR products with multiple sizes can be present, but should contain a product with the size of the left flank, antibiotic cassette and right flank combined, typically ~3,000 bp.
  9. Optional: Perform a MegaPrimer PCR with one the PCR product of one flank and the antibiotic resistance cassette to confirm that the desired MegaPrimer PCR product is not formed without all three components.
  10. Optional: Improve PCR yield by adding primers L1 and R1 (see Figure 1) to the PCR mix.
  11. Thaw 1 ml M-IV competent NTHi (protocol for making competent M-IV see Reference 2 and protocol in Notes below).
  12. Centrifuge 2 min at 10,000 x g in microcentrifuge.
  13. Remove the M-IV medium containing glycerol and resuspend the bacteria in 1 ml M-IV medium.
  14. Add target DNA and incubate 100 min. shaking with 100 rpm at 37 °C.
  15. Centrifuge 2 min at 10,000 x g in microcentrifuge.
  16. Remove 900 μl M-IV medium and plate 100 μl on selective BHI plates. Grow overnight at 37 °C + 5% CO2.
    Note: Record in your logbook the amount of colonies appearing on the selective plate.
  17. Pick four single colonies using a loop and streak them independently onto new selective BHI plates. Grow them overnight at 37 °C + 5% CO2.
    Note: From now on this protocol until step 23 is done for all 4 colonies from step 17.
  18. Resuspend colonies from step 17 in 5 ml of BHI medium with antibiotics.
  19. Grow bacteria until optical density at 620 nm = 0.2.
  20. Perform a chromosomal DNA isolation on the 5 ml culture from step 19 (e.g. CTAB protocol, for protocol see Notes below).
  21. Perform two control PCRs with isolated genomic DNA from step 20 to confirm the deletion of the target gene and the insertion of the antibiotic resistance cassette. Include as negative control the chromosomal DNA of the wild type strain.
    Reaction mix:
    2 μl 10x AmpliTaq PCR buffer (-MgCl2)
    2 μl 25 mM MgCl2*
    2 μl 2.5 mM dNTPs
    2 μl 4 pmol/μl primer L1
    2 μl 4 pmol/μl primer C (gene) or primer A (cassette)
    1 μl 5 ng/μl template chromosomal DNA
    0.2 μl AmpliTaq DNA polymerase
    8.8 μl dH2O
    Vtot = 20 μl
    PCR cycle:
    93 °C 4 min
    93 °C 30 sec     |
    55 °C 1 min      | 35x
    72 °C 1.5 min   |
    72 °C 4 min
    4 °C ∞
    *Optimize if necessary
  22. Load 2 μl PCR product mixed with DNA loading buffer on a 1.5% agarose gel with a 100 bp DNA marker.
  23. Check the gel for the size of the PCR product and select a mutant to continue with. Primer L1 + primer C (gene) should not result in an amplified PCR product (gene is removed), whereas the primer L1 + primer A (cassette) should give a PCR product (antibiotic cassette is inserted). Select the correct mutant and throw away all others.
  24. 1 µg chromosomal DNA of the mutant obtained in step 20 is 'crossed back' to the recipient wild type strain by transformation to prevent accumulation of (spontaneous) mutations because the chance that multiple mutations will transfer to the recipient bacterium is very low.
  25. Optional: To ensure that both mutant as wild-type strain have undergone the same (stress) conditions, also perform a transformation without adding DNA and without selection.
  26. Plate transformation mixture on (selective) BHI plates. Grow them overnight at 37 °C + 5% CO2.
  27. Optional: Make sure to dilute the wild type cells enough (e.g. 10,000 times) and plate onto a BHI agar plate without antibiotic. The mutant and equally treated wild type strain can be 'coupled' to for downstream experiments. It is possible to combine multiple mutants to one wild type strain.
  28. Pick a single colony from each plate using a loop and streak them independently onto new (selective) BHI plates. Grow them overnight at 37 °C + 5% CO2.
    Note: From now on this protocol is identical for the wild type and mutant strains.
  29. Resuspend colonies from step 28 in 5 ml of BHI medium with or without antibiotics.
  30. Grow bacteria until OD 0.2 at 620 nm and place on ice.
  31. Pipet 1.0 ml into a cryotube with 0.25 ml 80% glycerol and store at -80 °C.
  32. Use of the remaining media for chromosomal DNA isolation.
  33. Perform a chromosomal DNA isolation on the 5 ml culture from step 19 (e.g. CTAB protocol, for protocol see Notes below).
  34. Perform two control PCRs with isolated genomic DNA from step 33 with similar conditions as in step 21 to confirm the deletion of the target gene and the insertion of the antibiotic resistance cassette. Include as negative control the chromosomal DNA of the wild type strain.
  35. Load 2 μl PCR product mixed with DNA loading buffer on a 1.5% agarose gel with a 100 bp DNA marker.
  36. Check the gel for the size of the PCR product and select a mutant to continue with. Primer L1 + primer C (gene) should not result in an amplified PCR product, whereas the primer L1 + primer A (cassette) should give a PCR product. Select the correct mutant and throw away all others.

Notes

  1. Generation of M-IV competent NTHi (for details see Reference 2)
    1. Inoculate 10-15 ml sBHI medium in a 50 ml tube with a few colonies of an overnight sBHI plate and grow to an OD600 = 0.30-0.35 with 200 rpm at 37 °C.
    2. Centrifuge 10 min with 3,000 x g at room temperature.
    3. Aspirate the sBHI medium and remove the last amount of sBHI medium with a p1,000 pipet.
    4. Resuspend the bacteria in 1 ml PBS and add PBS to the start volume.
    5. Centrifuge 10 min with 3,000 x g at room temperature.
    6. Aspirate the PBS and remove the last amount of PBS with a pipet.
    7. Resuspend the bacteria in 1 ml M-IV medium and add M-IV medium to 10-15 ml (start volume).
    8. Incubate 100 min. with 100 rpm at 37 °C.
    9. Centrifuge 10 min with 3,000 x g at room temperature.
    10. Aspirate the M-IV medium and remove the last amount of M-IV medium with a pipet.
    11. Resuspend the bacteria in M-IV medium 10-15 ml (start volume).
    12. Optional: resuspend into 1 ml for NTHi with lower competence.
    13. Aliquot 1 ml of competent cells into tubes and add 0.25 ml 80% glycerol and store the bacteria at -80 °C.

  2. CTAB genomic DNA isolation protocol
    1. Inoculate 5 ml sBHI medium in a 50 ml tube with a few colonies of an overnight sBHI plate and grow to an OD600 = 0.30 - 0.35 with 200 rpm at 37 °C.
    2. Centrifuge 10 min with 3000 x g at room temperature.
    3. Aspirate the sBHI medium and resuspend the bacteria in 1 ml milli-Q water and transfer to a 2 ml tube.
    4. Add 70 µl 10% SDS (sodium dodecyl sulphate) and 5 µl 10 mg/ml proteinase K. Mix by inversion, do not vortex. Incubate at 65 °C for at least 10 min.
    5. Add 100 µl 5 M NaCl, mix and add 100 µl 10 % CTAB (N-Cetyl-N,N,N-trimethylammoniumbromid) in 0.7 M NaCl that was preheated at 65 °C. Vortex until the suspension turns white.
    6. Incubate at 65 °C for 10 min.
    7. Extract DNA by adding 500 µl of chloroform\isoamyl alcohol (24:1). Vortex for at least 10 sec.
    8. Centrifuge 5 min at max in a micro centrifuge.
    9. Transfer supernatant (~500 µl) to new micro centrifuge tube.
    10. Add 0.6 volume isopropanol (~300 µl) and mix well.
    11. Incubate at -20 °C for at least 30 min.
    12. Centrifuge 10 min at max in microcentrifuge.
    13. Carefully wash pellet with 500 µl 70% ethanol.
    14. Centrifuge 5 min at max in microcentrifuge.
    15. Carefully remove all liquid (if necessary, centrifuge shortly).
    16. Dry pellet on air (~10 min, pellet becomes white).
    17. Dissolve pellet in 100 µl Milli-Q water.

Acknowledgments

This protocol is adapted from a previously published paper: Langereis et al. (2013).

References

  1. Herriott, R. M., Meyer, E. M. and Vogt, M. (1970). Defined nongrowth media for stage II development of competence in Haemophilus influenzae. J Bacteriol 101(2): 517-524. 
  2. Langereis, J. D., Zomer, A., Stunnenberg, H. G., Burghout, P. and Hermans, P. W. (2013). Nontypeable Haemophilus influenzae carbonic anhydrase is important for environmental and intracellular survival. J Bacteriol 195(12): 2737-2746.

简介

在非典型流感嗜血杆菌中的定向缺失突变体可以通过靶基因与人工DNA构建体的等位基因交换来制备,其中抗生素抗性盒置于两个〜1000bp的DNA序列之间, 靶基因的5'和3'侧翼区。 该诱变所需的人工DNA构建体通过所谓的Megaprimer PCR方法合成(图1)。

关键字:流感嗜血杆菌, 基因缺失, 突变体, 诱变, 分子微生物学



图1. Megaprimer PCR方法的示意图。步骤A1。通过PCR(引物对L1 + L2和R1 + R2)扩增靶基因的侧翼区(〜1,000bp)。 L2和R2引物具有与抗生素抗性盒具有同源性的延伸。步骤A2。通过PCR扩增抗生素抗性盒(引物对L + R)。步骤B.组合第一PCR反应的三种PCR产物。侧翼区退火至抗生素抗性盒,形成一个大PCR产物。

材料和试剂

  1. 包括25mM MgCl 2和10×反应缓冲液(Applied Biosystems ,目录号:N8080171)的Amplitaq DNA聚合酶
  2. PWO DNA聚合酶,包括25mM MgSO 4和10×反应缓冲液(Roche Diagnostics,目录号:11644955001)。
  3. 10mM dNTP mix
  4. 无菌Milli-Q水
  5. QIAquick PCR纯化试剂盒(QIAGEN,目录号:28104)
  6. 琼脂糖
  7. 溴化乙锭
  8. 0.5x TBE
  9. 100bp的完美DNA梯(Merck Millipore,目录号:70539)
  10. 1kb完全DNA梯(Merck Millipore,目录号:70537)
  11. 1x PBS
  12. 细菌琼脂(BD Biosciences,目录号:212030)
  13. Bacto-Brain心浸液培养基(BD Biosciences,目录号:237500)
  14. 甘油
  15. M-IV感受态非典型流感嗜血杆菌(Herriott等人,1970),用于下面的方案。

设备

  1. T100热循环仪(Bio-Rad Laboratories)
  2. Nanodrop分光光度计(Thermo Fisher Scientific,Nanodrop,型号:ND1000)
  3. 离心机(Eppendorf,型号:5810)
  4. 微量离心机(Eppendorf,型号:5417R)
  5. Shaker(New Brunswick Scientific,型号:Innova 4000)
  6. 37℃,5%CO 2培养箱(BINDER GmbH,型号:CB 150)中。
  7. DNA凝胶电泳设备

程序

  1. PCR扩增抗生素抗性盒和靶基因的侧翼区。
    请参见图1,关于本PCR中使用的引物对,L1/L2,R1/R2和L/R 反应混合物:
    5μl10x PWO聚合酶缓冲液(-MgSO 4)
    3μl25mM MgSO 4 *
    5μl2.5 mM dNTPs
    5μl4 pmol /μl引物L1,R1或L,
    5μl4 pmol /μl引物L2,R2或R;
    1μl5ng /μl模板DNA *#
    0.5μlPWO DNA聚合酶
    25.5μldH 2 O
    Vtot = 50微升
    PCR循环:
    93℃-4分钟
    93°C 30秒  |
    55°C 1分钟    | 35x
    68℃1.5分钟|
    68℃5分钟
    12°C∞
    *如有必要,进行优化。 增加的MgSO 4或DNA模板浓度可能增加产量,但也可能降低特异性 侧翼区的模板是染色体DNA。 抗生素抗性盒的模板可以是抗性菌株的质粒或染色体DNA
  2. 用QIAquick PCR纯化试剂盒纯化PCR产物。
  3. 用Nanodrop测量DNA浓度。 > 20ng /μl的浓度足以用于Megaprimer PCR(步骤6)。
  4. 加载2μlPCR产物与DNA上样缓冲液混合在1.5%琼脂糖凝胶上,100 bp DNA标记。
  5. 检查凝胶的纯度(应为单个PCR产物)和产物的大小(大小取决于引物设计,通常〜1,000bp)。当所需产物未被扩增时,在步骤1中用修改的条件(例如,降低Tm,增加MgSO 4或添加DMSO)重复PCR。
  6. 进行MegaPrimer PCR以将靶基因的左侧和右侧侧翼区与抗生素抗性盒结合。
    反应混合物:
    5μl10x PWO聚合酶缓冲液(+ 20mM MgSO 4)
    5μl2.5 mM dNTPs
    〜200ng PCR产物L1 + L2(左侧区)
    〜200ng PCR产物R1 + R2(右侧区域) 〜400ng PCR产物R + L(抗生素抗性盒)
    0.5μlPWO DNA聚合酶
    Vtot =50μl,其中dH 2 O v/v PCR循环:
    93℃4分钟
    93°C 30秒|
    55°C 1分钟  | 30x
    68℃3分钟 |
    68℃7分钟
    12°C∞
  7. 加载2μlPCR产物与DNA加样缓冲液混合在0.7%琼脂糖凝胶上,用1 kb DNA标记。
  8. 检查凝胶的PCR产物的纯度和大小。 可以存在具有多种大小的PCR产物,但是应当含有具有左侧,抗生素盒和右侧组合的大小的产物,通常〜3,000bp。
  9. 可选:用一个侧翼的PCR产物和抗生素抗性盒进行MegaPrimer PCR,以确认没有所有三种组分都不形成所需的MegaPrimer PCR产物。
  10. 可选:通过向PCR混合物中加入引物L1和R1(见图1)来提高PCR产量。
  11. 解冻1 ml M-IV感受态NTHi(制备感受态M-IV的方案参见参考文献2和以下注释中的方案)。
  12. 在微量离心机中以10,000×g离心2分钟
  13. 取出含有甘油的M-IV培养基,并将细菌重悬在1ml M-IV培养基中
  14. 加入靶DNA,孵育100分钟。 在37℃下以100rpm振荡。
  15. 在微量离心机中以10,000×g离心2分钟
  16. 删除900微升M-IV培养基和板选择性BHI板上100微升。 在37℃+ 5%CO 2下生长过夜。
    注意:在您的日志中记录选择性平板上出现的菌落数量。
  17. 使用环挑选四个单菌落,并将它们独立地划线到新的选择性BHI平板上。 在37℃+ 5%CO 2下生长它们过夜。
    注意:从现在起,此协议将从步骤17的所有4个殖民地执行步骤23。
  18. 将来自步骤17的菌落重悬在5ml具有抗生素的BHI培养基中。
  19. 生长细菌直到620nm的光密度= 0.2。
  20. 在步骤19的5ml培养物上进行染色体DNA分离(例如,CTAB方案,方案参见下面的注释)。
  21. 使用来自步骤20的分离的基因组DNA进行两个对照PCR,以确认靶基因的缺失和抗生素抗性盒的插入。 包括野生型菌株的染色体DNA作为阴性对照。
    反应混合物:
    2μl10x AmpliTaq PCR缓冲液(-MgCl 2)
    2μl25mM MgCl 2 *
    2μl2.5 mM dNTPs
    2μl4 pmol /μl引物L1
    2微升4 pmol /μl引物C(基因)或引物A(盒)
    1μl5ng /μl模板染色体DNA
    0.2μlAmpliTaq DNA聚合酶
    8.8μldH 2 O
    Vtot = 20微升
    PCR循环:
    93℃4分钟
    93°C 30秒   |
    55°C 1分钟     | 35x
    72°C 1.5分钟   |
    72℃4分钟
    4℃∞
    *如有必要,优化
  22. 加载2μlPCR产物与DNA加样缓冲液混合在1.5%琼脂糖凝胶上,用100 bp DNA标记
  23. 检查凝胶的PCR产物的大小,并选择一个突变体继续。引物L1 +引物C(基因)不应产生扩增的PCR产物(除去基因),而引物L1 +引物A(盒)应当产生PCR产物(插入抗生素盒)。选择正确的突变体并丢弃所有其他突变体。
  24. 通过转化,将步骤20中获得的突变体的1μg染色体DNA"交叉回"到受体野生型菌株,以防止(自发)突变的累积,因为多个突变将转移到受体细菌的机会非常低。
  25. 可选:为了确保两个突变体作为野生型菌株已经经历相同(胁迫)条件,还进行转化而不添加DNA并且不进行选择。
  26. 板转化混合物在(选择性)BHI板上。在37℃+ 5%CO 2下生长它们过夜。
  27. 可选:确保足够稀释野生型细胞(例如 10,000次),并平板接种到没有抗生素的BHI琼脂平板上。突变体和同样处理的野生型菌株可以"偶联"用于下游实验。可以将多种突变体与一种野生型菌株组合。
  28. 使用环从每个板中挑选单个菌落,并将它们独立地划线到新的(选择性)BHI平板上。 在37℃+ 5%CO 2下生长它们过夜。
    注意:从现在开始,此方案对于野生型和突变株是相同的。
  29. 从步骤28重悬菌落在5ml含有或不含抗生素的BHI培养基中。
  30. 在620nm处生长细菌直至OD为0.2,并置于冰上。
  31. 将1.0ml吸管置于含有0.25ml 80%甘油的冷冻管中并储存在-80℃
  32. 使用剩余的培养基进行染色体DNA分离。
  33. 在步骤19的5ml培养物上进行染色体DNA分离(例如,CTAB方案,方案参见下面的注释)。
  34. 用来自步骤33的分离的基因组DNA用与步骤21类似的条件进行两个对照PCR,以确认靶基因的缺失和抗生素抗性盒的插入。 包括野生型菌株的染色体DNA作为阴性对照。
  35. 加载2μlPCR产物与DNA加样缓冲液混合在1.5%琼脂糖凝胶上,用100 bp DNA标记
  36. 检查凝胶的PCR产物的大小,并选择一个突变体继续。 引物L1 +引物C(基因)不应产生扩增的PCR产物,而引物L1 +引物A(盒)应产生PCR产物。 选择正确的突变体并丢弃所有其他突变体。

笔记

  1. M-IV感受态NTHi的产生(详见参考文献2)
    1. 在具有几个菌落的过夜sBHI平板的50ml管中接种10-15ml sBHI培养基,并在37℃下以200rpm生长至OD 600 = 0.30-0.35。
    2. 在室温下用3,000×g离心10分钟。
    3. 吸出sBHI培养基,并用p1000移液管除去最后一次的sBHI培养基
    4. 将细菌重悬在1ml PBS中,加入PBS至起始体积
    5. 在室温下用3,000×g离心10分钟。
    6. 吸出PBS,用移液管取出最后一滴PBS
    7. 将细菌重悬在1ml M-IV培养基中,加入M-IV培养基至10-15ml(起始体积)。
    8. 孵育100分钟。 在37℃下以100rpm转速
    9. 在室温下用3,000×g离心10分钟。
    10. 吸取M-IV培养基,用移液管取出最后一批M-IV培养基
    11. 重悬细菌在M-IV培养基10-15毫升(开始体积)。
    12. 可选:对于能力较弱的NTHi,重悬至1 ml
    13. 将1ml感受态细胞分装入管中,加入0.25ml 80%甘油,并将细菌保存在-80℃。

  2. CTAB基因组DNA分离方案
    1. 将5ml sBHI培养基接种在具有少量菌落的过夜sBHI平板的50ml管中,并在37℃下以200rpm生长至OD 600 = 0.30-0.35。
    2. 在室温下用3000×g离心10分钟。
    3. 吸出sBHI培养基并将细菌重悬于1ml milli-Q水中,并转移至2ml管。
    4. 加入70μl10%SDS(十二烷基硫酸钠)和5μl10mg/ml蛋白酶K.通过倒置混合,不要涡旋。 在65℃孵育至少10分钟。
    5. 加入100μl5M NaCl,混合并加入在65℃预热的0.7M NaCl中的100μl10%CTAB(N-十六烷基-N,N,N-三甲基溴化铵)。 旋转直到悬浮物变白。
    6. 在65℃孵育10分钟。
    7. 通过加入500微升氯仿\异戊醇(24:1)提取DNA。 涡旋至少10秒。
    8. 在微量离心机中最多离心5分钟
    9. 将上清液(约500μl)转移到新的微量离心管中
    10. 加入0.6体积异丙醇(〜300μl)并混匀
    11. -20℃孵育至少30分钟。
    12. 在微量离心机中最多离心10分钟
    13. 用500μl70%乙醇小心洗涤沉淀。
    14. 在微量离心机中最多离心5分钟
    15. 小心地取出所有液体(如果需要,立即离心)。
    16. 在空气中干燥沉淀(〜10分钟,沉淀变白)
    17. 将颗粒溶解在100μlMilli-Q水中

致谢

该协议改编自以前发表的论文:Langereis等人(2013)。

参考文献

  1. Herriott,R.M.,Meyer,E.M。和Vogt,M。(1970)。 为流感嗜血杆菌中的II期发展定义非传染性媒介。 J Bacteriol 101(2):517-524。 
  2. Langereis,J.D.,Zomer,A.,Stunnenberg,H.G.,Burghout,P.and Hermans,P.W。(2013)。 不可分型的流感嗜血杆菌碳酸酐酶对环境和细胞内存活很重要。/a> J Bacteriol 195(12):2737-2746。
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引用:Langereis, J. D. (2014). Generation of Non-typeable Haemophilus influenzae Directed Gene Deletion Mutants. Bio-protocol 4(5): e1066. DOI: 10.21769/BioProtoc.1066.
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