MAX Efficiency™ DH10Bac Competent Cells - FAQs

使用BaculoDirect表达试剂盒后，无法检测到任何重组融合蛋白。可能原因是什么？你们有何建议？

请检验入门克隆的构建，应确保插入片段位于载体读码框内。在LR反应后，利用PCR法分析重组病毒DNA，确认插入片段的大小和方向均正确。对PCR产物进行测序，确认用于表位标签表达的读码框正确。

我的P1病毒储液滴度较低，如何能够生成高滴度储液？

为得到高滴度储液，应使用P1储液再次感染细胞并生成P2高滴度储液。按照BaculoDirect使用手册第18页的说明，生成P2储液。

我进行了LR反应，并随后转染到Sf9/Sf21昆虫细胞中，但即使72小时后仍未观察到任何感染迹象。我该怎么办？

请查看以下建议：

•在转染到昆虫细胞中之前，利用PCR分析检验LR反应。
•在转染实验中，我们建议使用无添加的Grace’s昆虫细胞培养基代替无血清培养基，因为无血清培养基中的有些成分可能干扰转染。
•转染期间，应保证无FBS、无添加剂、无抗生素，因为这些物质中的蛋白质会干扰Cellfectin II试剂。
•LR重组反应中，以pENTR/CAT质粒为阳性对照，以Cellfectin II试剂（模拟转染）作为阴性对照。
•确保细胞处于对数生长期，存活率高于95%，细胞使用量应与使用手册的建议一致。
•若转染效率较低，细胞可能需要1周时间才能出现病毒感染迹象；继续培养，同时每日观察细胞的感染迹象。

更昔洛韦溶液中出现沉淀。我该怎么办？

在37°C水浴中温浴更昔洛韦5-10分钟，然后涡旋数分钟。沉淀应该重新溶解于溶液中。

我在使用His标签纯化柱对在昆虫细胞中表达的分泌型蛋白进行纯化时，未得到蛋白。为什么？我该怎么办？

用于培养昆虫细胞的培养基通常具有酸性pH（6.0-6.5）或含有电子供应基团，可阻止6xHis标签蛋白与Ni-NTA发生结合。昆虫细胞生长培养基所含谷氨酰胺、甘氨酸或组氨酸等氨基酸的浓度显著高于哺乳细胞生长培养基，这些氨基酸可与6xHis标签蛋白竞争Ni-NTA基质上的结合位点。例如，Grace’s培养基（Life Technologies）含有约10 mM谷氨酰胺、10 mM甘氨酸和15 mM组氨酸。

使用含适当成分和pH（8.0）的缓冲液对培养基进行透析，透析用缓冲液与在天然条件下进行纯化所使用的推荐裂解缓冲液相似，通常可恢复最佳的结合条件。应注意，选用的培养基可能导致出现白色沉淀（可能由不溶性盐组成），但6xHis标签蛋白通常留在溶液中。使用无蛋白质的培养基时，可通过蛋白质定量进行检测；或者利用免疫印迹分析法检测6xHis标签蛋白的含量。离心后，可从清澈的上清液中直接纯化6xHis标签蛋白。

从Bac-to-Bac杆状病毒表达系统得到的病毒储液一开始效果很好，但是几个月之后，蛋白表达明显降低。这是为什么？

请查看以下可能原因和解决方案：

•使用较高的起始MOI扩增病毒储液：使用低代数病毒储液，进行低MOI扩增。
•收获病毒上清液时，未离心和除去细胞：重新使用低代数病毒储液，进行低MOI扩增；如果该病毒储液代数为P2，则该储液可用于扩增。
•某些基因可使病毒变得非常不稳定：拿出分装的P2病毒储液，复苏后进行扩增。

使用Bac-to-Bac杆状病毒表达系统时，蛋白得率较低。可能原因是什么？

蛋白得率较低可能是因为：

•病毒储液含有重组和非重组杆状病毒的混合物：通过空斑纯化，分离重组杆状病毒。
•杆状病毒不是重组的：利用pUC/M13正向和反向引物对杆粒DNA进行PCR分析，对转座进行验证；使用新的重组杆粒DNA再次转染昆虫细胞。
•病毒滴度过低或过高：改变MOI。
•收获细胞的时间点不理想：采用针对表达的时间进程实验，确定获得最高蛋白表达水平的最佳时间。
•细胞生长条件和培养基不理想：根据培养瓶的尺寸和表达条件，优化细胞培养条件；为获得最佳的细胞生长和蛋白表达，我们建议使用Sf-900 II SFM或Sf-900 III SFM。
•所用细胞系不理想；尝试使用其他昆虫细胞系。
•细胞收获过晚：做一个时间进程实验，在不同时间点收获细胞。

我使用P2病毒储液感染细胞72小时后，未看到任何细胞感染迹象。这是为什么？

检查MOI。P1病毒的滴度低于估计值，可能导致MOI较低。

使用Bac-to-Bac杆状病毒表达系统，转染细胞后未看到任何病毒产生的迹象。已经5天了。这是为什么？该怎么解决？

请查看以下可能原因和建议：

•Cellfectin II试剂与杆粒没有混合或孵育时间不足：通过敲打或轻轻涡旋，混匀Cellfectin II试剂与杆粒，孵育混合物15-45分钟。
•杆粒得率低于预期：使用不同剂量的杆粒进行优化。
•杆粒在纯化或冻融过程中被剪切：在凝胶上验证杆粒的完整性。
•孵育时间不足：将混合物在27°C孵育8小时。
•所用细胞代数过高或已过对数生长期：为获得最佳结果，使用代数在8-15代之间的细胞；接种时，细胞应处于对数生长期。
•Cellfectin II试剂被冷冻过：购买新的试剂。
•所用培养基含有血清：转染时，使用无添加的Grace’s培养基。

已转染的细胞和只加了Cellfectin II试剂的阴性对照组细胞都在36小时内发生死亡。可能的原因是什么？

有多种可能原因：

•转染所用培养基含有抗生素。
•细胞接种密度过低：我们建议细胞汇合度至少达到70%。
•所用细胞代数过低：我们建议细胞用于转染前，至少生长5代。
•转染后未使用青霉素/链霉素，导致污染：加入转染混合物孵育5-8小时后，移除混合物，并在每孔中加入含有抗生素的培养基。

转染后细胞漂浮，看起来已死亡，但只加了Cellfectin II试剂的对照板是正常的。这是为什么？

这可能是因为来自杆粒制备的污染或细胞毒性。应设置无Cellfectin II试剂的杆粒阴性对照组。此外，在制备杆粒时，应使用PureLink HiPure质粒制备试剂盒，而不是基于硅胶的小量提取试剂盒。

我的杆粒DNA含有重组杆粒和空杆粒的混合物。哪里出错了？

最可能是挑选了中心为灰色或黑色的菌落。尝试分析更多的白色DH10Bac转化子。通常，我们建议挑选直径大于2 mm的白色菌落。将白色菌落在含50 µg/mL卡那霉素、7 µg/mL庆大霉素、10 µg/mL四环素、100 µg/mL Bluo-gal和40 µg/mL IPTG的新鲜的培养板上再次划线。孵育培养板24小时。

我对重组杆粒进行PCR分析，但未得到产物。有哪些可能原因和建议？

以下是针对该问题的可能原因和建议：

•使用纯的校正聚合酶进行分析：使用Taq聚合酶进行分析。
•插入片段太长，导致PCR出现问题：使用与您的M13引物成对的基因特异性引物，代替使用M13正向和反向引物。
•在目的基因中，富含GC长片段：在PCR反应中，考虑使用DMSO（高达8%）。

我的杆粒DNA发生了降解。有哪些可能原因和建议？

请查看以下可能原因和建议：

•DNA保存不恰当：为避免反复冻融，应将纯化的杆粒DNA分装保存在–20°C。
•对高分子量杆粒DNA的处理不恰当：在分离杆粒DNA时，不要涡旋振荡DNA溶液；此外，不要机械地重悬DNA沉淀；将溶液置于管内，轻轻敲打几次。

我尝试分离杆粒DNA，但过夜培养后并未生长。这是为什么？

这可能是因为：

•使用了错误的抗生素或旧的培养基：应使用新鲜的培养基。
•菌落太老或太小：从新划线的培养板上选取较大的白色菌落。
•目的基因的特殊性质导致插入片段不稳定；例如，直接重复序列：应在30°C培养24小时，而不是在37°C培养过夜。

我在使用Bac-to-Bac表达系统生成重组杆粒DNA时，蓝/白菌落区别很小。我该怎么办？

菌落的颜色区别很小可能是因为：

•琼脂的pH不正确：调整LB琼脂的pH至7.0。
•蓝色强度太弱；应确保您使用的是Bluo-gal，而不是X-Gal。您也可尝试将Bluo-gal的浓度增加至300 µg/mL。
•培养板上的菌落过多或过少：调整细胞的连续稀释度，从而获得最佳的菌落数量。
•孵育时间过短或温度过低：接种后48小时，方可挑选菌落；在37°C孵育培养板。
•IPTG浓度不理想：IPTG浓度范围在20–60 µg/mL之间，通常可得到最佳的颜色区分。

我转染了杆粒DNA，却未看到感染迹象。我应如何对重组杆粒DNA进行分析？

我们建议取40μL样品的八分之一在0.5% TAE琼脂糖凝胶上电泳。以23伏缓慢电泳12小时。中提的重组bacmid的条带形态应该可以看见。

我在使用Bac-to-Bac表达系统生成重组杆粒DNA时，得到的菌落非常少。可能原因和解决方案是什么？

请查看以下可能原因和建议：

•复苏/表达期间使用了LB培养基：在4小时的生长期使用SOC培养基。
•复苏/表达时间过短：延长复苏时间，在37°C时为4小时以上，或在30°C时为6小时。
•IPTG浓度不理想：我们建议使用20–40 µg/mL IPTG。

我在使用Bac-to-Bac表达系统生成重组杆粒DNA时，得到的所有菌落均为蓝色。可能原因和解决方案是什么？

请查看以下可能原因和建议：

•用于转化的pFastBac DNA质量较差：使用纯化的质粒DNA进行转化，检查质粒DNA的质量。
•培养板中缺少庆大霉素：制备含50 µg/mL卡那霉素、7 µg/mL庆大霉素、10 µg/mL四环素、100 µg/mL Bluo-gal和40 µg/mL IPTG的新鲜的选择性培养板。

我在使用Bac-to-Bac表达系统生成重组杆粒DNA时，得到的所有菌落均为白色。我是否应预期看到一些蓝色菌落？

尽管您将要挑选的是白色（重组）菌落，但是您也应该预期会看到一些蓝色（含非重组杆粒）菌落。以下是关于无蓝色菌落的可能原因和建议：

•颜色形成时间不足：在鉴定菌落表型前，至少等待48小时。
•在琼脂板中，使用Bluo-gal代替X-Gal：在板中使用Bluo-gal，可增强蓝白菌落的对比度。
•转座后，生长不充分：接种前，使转化后细胞在SOC培养基中生长至少4小时。
•培养板中缺少Bluo-gal和IPTG：制备含50 µg/mL卡那霉素、7 µg/mL庆大霉素、10 µg/mL四环素、100 µg/mL Bluo-gal和40 µg/mL IPTG的新鲜的选择性培养板。
•培养板上菌落过多：连续稀释转化混合物，从而获得间隔合适的菌落（建议稀释度为10-2至10-4）。
•培养板太久或在光照下保存：不要使用放置超过4周的培养板；培养板应避光保存。
•孵育时间过短或温度过低：挑选菌落前，至少等待48小时。在37°C孵育培养板。

Bac-N-Blue表达系统与Bac-to-Bac表达系统有何区别？

在Bac-N-Blue系统中，转移载体和杆状病毒DNA的重组发生于昆虫细胞中。Bac-N-Blue载体是一种线性AcMNPV衍生物，含有不完整的(3’) lacZ片段。相应的转移载体含有一个5’ lacZ片段。在同源重组的基础上，重组Bac-N-Blue杆状病毒DNA将具有一个受PETL启动子控制的lacZ基因。因此，重组Bac-N-Blue杆状病毒在空斑实验中会形成蓝色空斑，易于识别。在Bac-to-Bac表达系统中，转移载体与杆状病毒DNA间的重组或位点特异性转座发生于大肠杆菌（DH10Bac）中。在Bac-to-Bac表达系统中，含重组杆状病毒DNA的菌落筛选发生于含50 μg/mL卡那霉素（杆状病毒质粒）、7 μg/mL庆大霉素（pFastBac）、10 μg/mL四环素（辅助性质粒）、100 μg/mL Bluo-gal和40 μg/mL IPTG的Luria琼脂板上。

为防止杆状病毒表达系统中发生蛋白质水解作用，建议采取哪些措施？

以下是关于如何防止杆状病毒表达系统中发生蛋白质水解作用的一篇很好的文献：
Hom LG, Volkman LE (1998) Preventing proteolytic artifacts in the Baculovirus expression system. BioTechniques 25:18–20.

杆状病毒是否有包装极限？

杆状病毒的杆会根据包装DNA的需要而持续延伸。因此，理论上，该系统能够容纳几百个Kb。在包装极限成为问题之前，标准克隆技术将限制插入片段大小。

杆状病毒是否有首选的分泌信号？

昆虫信号肽和/或前序列无法总是增强杆状病毒系统中外源分泌途径蛋白的表达和/或分泌。请查看以下文献：

•Jarvis DL, Summers MD, Garcia A Jr, Bohlmeyer DA (1993) Influence of different signal peptides and prosequences on expression and secretion of human tissue plasminogen activator in the baculovirus system. J Biol Chem 268(22):16754–16762.
•Tessier DC, Thomas DY, Khouri HE, Laliberte F, Vernet T (1991) Secretion of a plant protein in the baculovirus system was enhanced when its signal peptide was replaced with an insect-derived signal peptide. Gene (Amst.) 98:177–183.

我想表达较大的蛋白，分子量高于130 kDa。能否使用杆状病毒系统或者蛋白是否会降解？

我们的研发团队已成功表达了分子量高达300 kDa的蛋白。如果在血清含量>2%的培养基中表达，可将降解程度降至最低。如果您不介意采用额外的纯化步骤，可使用10%血清。为了确定获得最佳表达所需的最短收获时间，我们强烈推荐采用感染复数为5-10的高滴度储液进行不同时间的感染。每24小时为一个时间点，持续5天。

杆状病毒多角体蛋白的大小是多少？

多角体蛋白为30 kDa。

Bac-to-Bac表达系统使用的启动子是什么？

驱动目的基因的启动子是多角体启动子。尽管对于大多数蛋白，多角体启动子通常强3-5倍，但该启动子可被p10启动子取代。然而，经高度修饰或分泌的蛋白在p10启动子的驱动下可达到更高的表达效率，因为p10启动子在末期阶段的早期激活，而多角体启动子在非常晚的末期才激活，两者正好相反。蛋白复合物高效和正确加工所需的细胞蛋白合成，在非常晚的末期停止。这解释了为什么一些报告提到在分泌和修饰蛋白的表达中，p10启动子与多角体启动子同样有效或p10启动子比多角体启动子的效率高2倍。但是，在大多数情况下，多角体启动子也有效。至于哪种启动子更强，取决于多种因素，包括蛋白质的性质和感染后的收获时间。

你们提供Subcloning Efficiency、Library Efficiency和MAX Efficiency感受态细胞。它们的区别是什么？

有少数例外，但他们主要的区别在于所保证的转化效率：

Subcloning Efficiency细胞每转化1 µg pUC19或pUC18超螺旋质粒保证产生至少1.0 x 10E6个转化子。
Library Efficiency细胞每转化1 µg pUC19或pUC18 DNA保证产生至少1.0 x 10E8个转化子。
MAX Efficiency细胞每转化1 µg pUC19或pUC18 DNA保证产生至少1.0 x 10E9个转化子。

I cannot detect any recombinant fusion protein after using the BaculoDirect Expression Kit. What could be the cause for this and what do you suggest I try?

Please check the construction of your entry clone, and ensure that the insert is in frame with the vector. Analyze the recombinant viral DNA by PCR to confirm the correct size and orientation of your insert after the LR reaction. Sequence your PCR product to verify the proper reading frame for expression of the epitope tag.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I'm getting a low-titer P1 viral stock and would like to generate a high-titer stock. What should I do?

To get a high-titer stock, reinfect cells with the P1 stock and generate a P2 high-titer stock. Follow the directions in the BaculoDirect manual on page 18 to generate your P2 stock.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I performed an LR reaction, followed by transfection into Sf9/Sf21 insect cells, but do not see any signs of infection even though it's been 72 hours. What should i do?

Please see our recommendations below:

- Check the LR reaction by PCR analysis prior to transfection into insect cells.
- We recommend using Grace's Insect Cell Culture Medium, Unsupplemented during the transfection experiment instead of serum-free medium, as components in serum-free medium may interfere with transfection.
- Ensure that FBS, supplements, or antibiotics are not included during transfection, as the proteins in these materials can interfere with the Cellfectin II Reagent.
- Use the LR recombination reaction using the pENTR/CAT plasmid as a positive control and Cellfectin II Reagent only (mock transfection) as a negative control.
- Ensure that cells are in the log phase of growth with >95% viability, and the amount of cells are in accordance with the suggestions in the manual.
- Cells may not show signs of viral infection for up to a week depending on transfection efficiency; continue culturing and monitor cells daily for signs of infection.

I see a precipitate in my ganciclovir solution. What can I do?

Warm the ganciclovir solution in a water bath at 37 degrees C for 5-10 min, then vortex for a few minutes. The precipitate should go back into solution.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I am purifying my secreted protein expressed in insect cells with a His-tagged purification column and getting no yield of my protein. Why and what can I do?

Media used to culture insect cells usually have an acidic pH (6.0-6.5) or contain electron-donating groups that can prevent binding of the 6xHis-tagged protein to Ni-NTA. Amino acids such as glutamine, glycine, or histidine are present at significantly higher concentrations in media for growing insect cells than in media for growing mammalian cells, and compete with the 6xHis-tagged protein for binding sites on Ni-NTA matrices. Grace's medium (Thermo Fisher Scientific), for example, contains approximately 10 mM glutamine, 10 mM glycine, and 15 mM histidine.

Dialysis of the medium against a buffer with the appropriate composition and pH (8.0) similar to the lysis buffer recommended for purification under native conditions usually restores optimal binding conditions. Note that depending on the medium used, a white precipitate (probably made up of insoluble salts) can occur, but normally the 6xHis-tagged protein remains in solution. This can be tested by either protein quantitation if using a protein-free medium or by monitoring the amount of 6xHis-tagged protein by western-blot analysis. After centrifugation, 6xHis-tagged protein can be directly purified from the cleared supernatant.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

My viral stock used to work well from my Bac-to-Bac baculovirus expression system, but after a couple of months, there is much less protein expression. Why is this?

Please review the following possibilities and solutions:

- Viral stock was amplified using high MOI originally: Go back to the lower-passage viral stock and do a low-MOI amplification.
- Did not spin down and get rid of cells when harvesting viral supernatant: Go back to the lower-passage viral stock and do a low-MOI amplification; if this viral stock is P2, this stock can be used in amplification.
- For some genes, the virus can become very unstable: Free the aliquoted P2 viral stock and do one run of amplification after reviving.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I'm getting low protein yield when using the Bac-to-Bac baculovirus expression system. What are the possible reasons for this?

Low protein yield may occur due to the following reasons:

- Viral stock contains a mixture of recombinant and non-recombinant baculovirus: Perform plaque purification to isolate recombinant baculovirus.
- Baculovirus is not recombinant: Verify transposition by PCR analysis of bacmid DNA using pUC/M13 forward and reverse primers; re-transfect insect cells with new recombinant bacmid DNA.
- Use too low or too high viral titer: Vary the MOI.
- Time of cell harvest is not optimal: perform a time course of expression to determine the optimal time to obtain maximal protein expression.
- Cell growth conditions and medium are not optimal: Optimize cell culture conditions based on the size of your culture vessel and expression conditions; we recommend using Sf-900 II SFM or Sf-900 III SFM for optimal cell growth and protein expression.
- Cell line is not optimal; try other insect cell lines.
- Cells were harvested too late: Do a time-course experiment and harvest cells at different time points.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I don't see any signs of cell infection 72 hours after infection with my P2 viral stock. Why is this?

Check the MOI. It may be low because the titer of the P1 virus is lower than what was estimated.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Using the Bac-to-Bac baculovirus expression system, I've transduced my cells but do not see any signs of viral production. It has been 5 days. What happened and what should I do?

Please see the possible reasons and suggestions below:

- Mixture of Cellfectin II Reagent and bacmid was not performed or was not incubated long enough: Mix the Cellfectin II Reagent and bacmid well by tapping or gentle vortexing, and incubate the mixture for 15-45 min.
- Bacmid yield is lower than estimated: Set up an optimization with different amounts of bacmid.
- Bacmid is sheared during purification or freeze/thaw: Verify the integrity of bacmid on a gel.
- Incubation time is not long enough: Incubate mix for 8 hr at 27 degrees C.
- Cells used are of high passages or have passed log-phase growth: For best results, use cells between 8-15 passages; plate cells when they are in log-phase growth.
- Cellfectin II Reagent has been frozen: Purchase a new vial.
- Medium used contains serum: Use unsupplemented Grace's medium in transfection.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Both transfected cells and Cellfectin II Reagent alone negative control cells are dead within 36 hours. What could cause this?

There are several possibilities:

- Using media containing antibiotics during transfections.
- Plating cells at too low a density: We recommend at least 70% confluence.
- Using cells at too early a passage: We recommend growing cells for at least 5 passages before using them for transfection.
- Contamination because of no pen/strep after the transfection: After 5-8 hr incubation with the transfection mixture, remove the mixture and add antibiotics containing media/well.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

My transfected cells are floating up and look dead, but the Cellfectin II Reagent alone control plate is fine. What could be happening?

This may be due to contamination or cytotoxicity from the bacmid prep. Make sure to include a negative control that is the bacmid only without Cellfectin II Reagent. Additionally, use the PureLink HiPure plasmid prep kit, not the silica-based miniprep kit for bacmid prep.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

My bacmid DNA contains a mixture of recombinant bacmid and empty bacmid. What am I doing wrong?

Most likely, a colony that was gray or dark in the center was picked. Try to analyze more white DH10Bac transformants. Typically, we recommend picking a white colony whose diameter is >2 mm. Restreak the white colonies on a fresh plate with 50 µg/mL kanamycin, 7 µg/mL gentamicin, 10 µg/mL tetracycline, 100 µg/mL Bluo-gal and 40 µg/mL IPTG. Incubate plates for 24 hours.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

My PCR analysis of the recombinant bacmid gives no product. What could be the cause of this and do you have any recommendations on how to fix this?

Please see the possible causes and suggestions we have to alleviate this problem:

- Insert is very long and causes difficulties in PCR: Instead of using both M13 forward and reverse primers, use one gene-specific primer paired with the M13 primer of your choice.
- Long GC-rich stretches in the gene of interest: Consider using DMSO (up to 8%) in the PCR reaction.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

My bacmid DNA is degraded. What could be the cause for this and what recommendations do you have to fix this?

Please see the possible reasons and suggestions below:

- DNA stored improperly: Ensure that purified bacmid DNA is stored at -20 degrees C in aliquots to avoid repeated free/thaws.
- High molecular weight bacmid DNA handled improperly: When isolating bacmid DNA, do not vortex the DNA solution; additionally, do not resuspend DNA pellets mechanically; allow solution to sit in the tube with occasional tapping.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I am trying to isolate my bacmid DNA but the overnight culture did not grow. What could have happened?

This could be caused by the following:

- Wrong antibiotic or old media: use fresh media.
- Colonies are too old or too small: Use large white colonies from freshly streaked plates.
- Unstable insert caused by special feature of the gene of interest; for example, direct repeats: Incubate the culture at 30 degrees C for 24 hours instead of 37 degrees C overnight.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I'm trying to generate recombinant bacmid DNA using the Bac-to-Bac expression system and am seeing very poor blue/white colony differentiation. What should I do?

Poor color differentiation for your colonies could be caused by the following:

- Agar is not at the correct pH: Adjust pH of LB agar to 7.0.
- Intensity of the blue color is too weak; ensure that you are using Bluo-gal, not X-Gal. You can also try increasing the concentration of Bluo-gal to 300 µg/mL.
- Too many or too few colonies on the plate: Adjust the serial dilutions of cells to obtain an optimal number of colonies.
- Incubation period too short or temperature too low: Do not pick colonies until 48 hours after plating; incubate plates at 37 degrees C.
- IPTG concentration is not optimal: A range of 20-60 µg/mL IPTG generally gives optimal color development.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I transfected my bacmid DNA but saw no signs of infection. The quality of my DNA prep might be an issue. How can I analyze the bacmid DNA?

We recommend running 1/8th of the 40 µL midiprep sample on a 0.5% TAE agarose gel. Electrophorese slowly at 23 V for 12 hr. The banding pattern of the recombinant bacmid midiprep should be seen.

I'm trying to generate recombinant bacmid DNA using the Bac-to-Bac expression system and I'm getting very few colonies. What could be the cause for this and do you have any recommendations for how to fix this?

Please review the following reasons and our recommendations:

- Use LB medium for recovery/expression period: Use SOC medium for the 4 hr growth time.
- Recovery/expression time too short: Increase the recovery time to > 4 hr at 37 degrees C or 6 hr at 30 degrees C.
- IPTG concentration is not optimal: We suggest using 20-40 µg/mL IPTG.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I'm trying to generate recombinant bacmid DNA using the Bac-to-Bac expression system and all of my colonies are blue. What could be the cause of this and what should I do?

Please review the following possibilities and recommendations:

- pFastBac DNA used for transformation was of poor quality: Use purified plasmid DNA for transformation and check the quality of your plasmid DNA.
- Gentamicin omitted from plates: Prepare fresh selective plates containing 50 µg/mL kanamycin, 7 µg/mL gentamicin, 10 µg/mL tetracycline, 100 µg/mL Bluo-gal, and 40 µg/mL IPTG.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I'm trying to generate recombinant bacmid DNA using the Bac-to-Bac expression system and all the colonies obtained are white. Shouldn't I expect to see some blue colonies?

Although you will be picking white (recombinant) colonies, you should expect to see some blue (contain non-recombinant bacmid) colonies. Here are some possible causes for seeing no blue colonies and recommendations for the same:

- Insufficient time for color development: Wait at least 48 hours before identifying colony phenotypes.
- Use Bluo-gal instead of X-Gal in agar plates: Use Bluo-gal in plates to increase contrast between blue and white colonies.
- Insufficient growth after transposition: Grow transformed cells in SOC medium for a minimum of 4 hours before plating.
- Bluo-gal and IPTG omitted from plates: Prepare fresh selective plates containing 50 µg/mL kanamycin, 7 µg/mL gentamicin, 10 µg/mL tetracycline, 100 µg/mL Bluo-gal, and 40 µg/mL IPTG.
- There are too many colonies on the plate: Serially dilute the transformation mix to obtain well-spaced colonies (10-2 to 10-4 is suggested).
- Plates are too old or stored in light: Do not use plates that are more than 4 weeks old; store plates protected from light.
- Incubation period too short or temperature is too low: Wait at least 48 hours before picking colonies. Incubate plates at 37 degrees C.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

What is the difference between the Bac-N-Blue expression system and the Bac-to-Bac expression system?

In the Bac-N-Blue system, recombination between the transfer vector and the baculovirus DNA occurs in insect cells. The Bac-N-Blue vector is a linearized AcMNPV derivative that contains an incomplete (3') lacZ fragment. The corresponding transfer vector contains a 5' lacZ fragment. Upon homologous recombination, the recombinant Bac-N-Blue baculovirus DNA will have a complete lacZ gene that is under the control of the PETL promoter. Thus, recombinant Bac-N-Blue baculovirus will provide blue plaques in the plaque assay and can be easily identified. In the Bac-to-Bac expression system, recombination or site-specific transposition between transfer and baculovirus DNA occurs in E. coli (DH10Bac). In the Bac-to-Bac expression system, selection of colonies containing recombinant baculovirus DNA occurs in the presence of Luria Agar plates with 50 µg/mL kanamycin (bacmid), 7 µg/mL gentamycin (pFastBac), 10 µg/mL tetracycline (helper plasmid), 100 µg/mL Bluo-gal, and 40 µg/mL IPTG.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

What steps are recommended for preventing proteolysis in the baculovirus expression systems?

The following is an excellent reference for how to prevent proteolytic artifacts in the baculovirus expression system:
Hom LG, Volkman LE (1998) Preventing proteolytic artifacts in the Baculovirus expression system. BioTechniques 25:18-20.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Is there a packaging limit for the baculovirus?

The baculovirus rod will continue to elongate as required to package the DNA. Thus, the system could theoretically accommodate hundreds of Kb. Standard cloning techniques will limit the insert size before packaging limits become an issue.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Are there preferred secretion signals for use with baculovirus?

Insect-derived signal peptides and/or prosequences cannot always enhance the expression and/or secretion of foreign secretory pathway proteins in the baculovirus system. Please see the following references:

- Jarvis DL, Summers MD, Garcia A Jr, Bohlmeyer DA (1993) Influence of different signal peptides and prosequences on expression and secretion of human tissue plasminogen activator in the baculovirus system. J Biol Chem 268(22):16754-16762.
- Tessier DC, Thomas DY, Khouri HE, Laliberte F, Vernet T (1991) Secretion of a plant protein in the baculovirus system was enhanced when its signal peptide was replaced with an insect-derived signal peptide. Gene (Amst.) 98:177-183.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I'm interested in expressing a large protein, larger than 130 kDa. Can I use the baculovirus system or will the proteins be degraded?

Our R&D team has successfully expressed proteins up to 300 kDa. If they express in >2% serum, it should minimize degradation. If you don't mind the extra step of purification, 10% serum could be used. We highly recommend doing a time-course infection with high-titer stock, with a MOI of 5-10, to make an assessment of the minimum harvesting time necessary for the best expression. Time points should be taken every 24 hours for 5 days.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

What is the size of the baculovirus polyhedron protein?

The polyhedron protein is 30 kDa.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

What is the promoter used in the Bac-to-Bac expression system?

The promoter that drives the gene of interest is the polyhedron promoter.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

How do you recommend that I prepare my DNA for successful electroporation of E. coli?

For best results, DNA used in electroporation must have a very low ionic strength and a high resistance. A high-salt DNA sample may be purified by either ethanol precipitation or dialysis.

The following suggested protocols are for ligation reactions of 20ul. The volumes may be adjusted to suit the amount being prepared.

Purifying DNA by Precipitation: Add 5 to 10 ug of tRNA to a 20ul ligation reaction. Adjust the solution to 2.5 M in ammonium acetate using a 7.5 M ammonium acetate stock solution. Mix well. Add two volumes of 100 % ethanol. Centrifuge at 12,000 x g for 15 min at 4C. Remove the supernatant with a micropipet. Wash the pellet with 60ul of 70% ethanol. Centrifuge at 12,000 x g for 15 min at room temperature. Remove the supernatant with a micropipet. Air dry the pellet. Resuspend the DNA in 0.5X TE buffer [5 mM Tris-HCl, 0.5 mM EDTA (pH 7.5)] to a concentration of 10 ng/ul of DNA. Use 1 ul per transformation of 20 ul of cell suspension.

Purifying DNA by Microdialysis: Float a Millipore filter, type VS 0.025 um, on a pool of 0.5X TE buffer (or 10% glycerol) in a small plastic container. Place 20ul of the DNA solution as a drop on top of the filter. Incubate at room temperature for several hours. Withdraw the DNA drop from the filter and place it in a polypropylene microcentrifuge tube. Use 1ul of this DNA for each electrotransformation reaction.

You offer competent cells in Subcloning Efficiency, Library Efficiency and MAX Efficiency. How do these differ?

There are a few exceptions, but in general the difference is in guaranteed transformation efficiency as follows:

Subcloning Efficiency cells are guaranteed to produce at least 1.0 x 10E6 transformants per µg of transformed pUC19 or pUC18 supercoiled plasmid
Library Efficiency cells are guaranteed to produce at least 1.0 x 10E8 transformants per µg pUC19 or pUC18 DNA
MAX Efficiency cells are guaranteed to produce at least 1.0 x 10E9 transformants per µg pUC19 or pUC18 DNA

When should DMSO, formamide, glycerol and other cosolvents be used in PCR?

Cosolvents may be used when there is a failure of amplification, either because the template contains stable hairpin-loops or the region of amplification is GC-rich. Keep in mind that all of these cosolvents have the effect of lowering enzyme activity, which will decrease amplification yield. For more information see P Landre et al (1995). The use of co-solvents to enhance amplification by the polymerase chain reaction. In: PCR Strategies, edited by MA Innis, DH Gelfand, JJ Sninsky. Academic Press, San Diego, CA, pp. 3-16.

Additionally, when amplifying very long PCR fragments (greater than 5 kb) the use of cosolvents is often recommended to help compensate for the increased melting temperature of these fragments.

Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.