轉(zhuǎn)染實驗技巧（如下信息全部由客戶提供）

DNA體外轉(zhuǎn)染試劑

如何準備轉(zhuǎn)染所需的質(zhì)粒DNA

轉(zhuǎn)染效率受到諸多因素的影響，除了細胞、培養(yǎng)基和載體等影響因素外，另外一項非常重要的因素便是DNA的質(zhì)量。為了比較不同廠家的轉(zhuǎn)染效率，我們分別從三家不同的供應商購買了質(zhì)粒制備試劑盒來制備pEGFP-N3質(zhì)粒DNA，并通過不同的方法將制備的pEGFP-N3質(zhì)粒轉(zhuǎn)運至NIH-3T3細胞。

pEGFP-N3質(zhì)粒分別通過Endofree Maxi Piasmid Kit(Qiagen),PerfectPrep Endofree Piasmid Maxi Kit(5 PRIME,inc)及NucleoBond DNA Maxi Kit(MACHEREY-NAGEL GmbH&Co.KG)制備，我們測定了230,260,280及320nm的吸光值檢測DNA的質(zhì)量。通過上述三種方法制備的DNA質(zhì)量如下：MACHEREY-NAGEL＞5Prime＞Qiagen。

pEGFP-N3由PolyJetDNA轉(zhuǎn)染試劑運至NIH-3T3細胞。轉(zhuǎn)染效率的高低與DNA的質(zhì)量相符，由MACHEREY-NAGEL純化DNA對應的轉(zhuǎn)染效率最高，而由Qiagen純化DNA對應的轉(zhuǎn)染效率最低。因此，MACHEREY-NAGEL GmbH & Co.KG的質(zhì)粒制備試劑盒被認為是制備轉(zhuǎn)染級別DNA的最佳選擇。

How to prepare transfection grade plasmid DNA

Transfection efficiencies are affected by a variety of different parameters.  Besides factor such as cell culture, medium and vectors, one of most critical parameters is DNA quality.  We prepared pEGF-N3 plasmid DNA by large plasmid preparation kits from three different vendors and tested transfection efficiencies by delivering pEGFP-N3 DNAs prepared by different methods to NIH-3T3 cells.  

We prepared pEGFP-N3 plasmid with Endofree Maxi Plasmid Kit (Qiagen),  PerfectPrep Endofree Plasmid Maxi Kit (5 PRIME, Inc.) and NucleoBond DNA Maxi Kit (MACHEREY-NAGEL GmbH & Co. KG).  The DNA quality was determined by measuring absorbance at 230, 260, 280 and 320 nm by spectrometry.
The purity of DNA prepared by the three methods is as follows:
MACHEREY-NAGEL > 5 Prime > Qiagen. 

Then pEGFP DNA was delivered with PolyJet™ (Cat # SL100688) DNA transfection reagent to NIH-3T3 cells per manufacturer’s protocol.  In accordance with DNA purity results, we found that DNA from MACHEREY-NAGEL gave the best transfection efficiency while Qiagen DNA gave the worst efficiency.  Therefore plasmid preparation kit from MACHEREY-NAGEL GmbH & Co. KG is confirmed to be the best choice to prepare tansfection grade DNA.

表皮細胞的轉(zhuǎn)染

表皮細胞廣泛遍布于身體，正常的表皮細胞較難轉(zhuǎn)染，尤其是使用基于脂質(zhì)體技術的轉(zhuǎn)染試劑。我們使用電轉(zhuǎn)(Amaxa)方法轉(zhuǎn)染正常人的結(jié)腸表皮細胞并得到了65%的GFP標記細胞。非常感謝SignaGen,現(xiàn)在我們使用GenJet Ver II可以成功轉(zhuǎn)染正常人的結(jié)腸表皮細胞并且轉(zhuǎn)染效率顯著提高至75%。

如下是使用GenJet Ver II(Cat#SL100489)轉(zhuǎn)染表皮細胞的簡單步驟：

1、轉(zhuǎn)染時確保表皮細胞達到85%的融合度，并且保證細胞新鮮、健康。

2、對于6孔板，用不含血清的DMEM分別稀釋1.0µg,DNA及3.0µl,GenJet Ver.II（Cat＝SL100489）.將稀釋好的轉(zhuǎn)染試劑加入DNA中，室溫下放置15分鐘以形成轉(zhuǎn)染復合物。

3、將轉(zhuǎn)染復合物直接加入表層細胞中：6孔板，每孔含1.0ml培養(yǎng)基，在血清/抗生素存在下，轉(zhuǎn)染進行。

4、在轉(zhuǎn)染進行5小時后，清除轉(zhuǎn)染復合物并換成正常生長培養(yǎng)基。

5、轉(zhuǎn)染后的24-48小時，檢測轉(zhuǎn)染基因的表達情況。

Transfection of epithelial cells.

Epithelial cells are found throughout the body, from skin to glandular formations within tissues. In vivo these cells are attached to a three dimensional basement membrane matrix. Normal epithelial cell is extremely hard to transfect especially to liposome based transfection reagents. We ever used electroporation (Amaxa) to transfect normal human colonic epithelial cells and got 65% GFP+ cells. Thanks to SignaGen, now we have successfully transfected normal human colonic epithelial cell with GenJet Ver. II with up to 75% GFP+ efficiency. The following is a brief protocol for transfecting epithelial cell with GenJet Ver. II (Cat # SL100489):

1. Grow epithelial cell ~85% confluency at time of transfection. Epithelial cells must freshly prepared and healthy. 

2. For 6-well plate, dilute 1.0 µg of DNA and 3.0 µl of GenJet Ver. II (Cat # SL100489) per well with serum free DMEM respectively. Add diluted reagent to diluted DNA and let transfection complex formed at RT for 15 minutes.

3. Add transfection complex to epithelial cells directly. Transfection is conducted in presence of serum/antibiotics with transfection volume of 1.0 ml per well of 6-well plate.

4. Remove transfection complex 5 hours after transfection and change back to normal growth medium.

5. Check transgene expression 24~48 hours post transfection.

針對特定細胞選擇更有效的實驗步驟

GenJet Ver.II，LipoD293及PolyJet針對哺乳動物細胞的DNA體外轉(zhuǎn)染試劑，均為您提供兩種不同的轉(zhuǎn)染步驟——一般步驟及高級步驟，這兩種步驟分別針對不同的哺乳動物細胞。高級步驟主要針對難轉(zhuǎn)的哺乳動物細胞，例如：MDCK，MDA-MB231，Caco-2等細胞。使用一般的轉(zhuǎn)染步驟若是轉(zhuǎn)染效率低于5%，那么使用高級轉(zhuǎn)染步驟則可以將轉(zhuǎn)染效率提升至60%。

如何選擇轉(zhuǎn)染步驟則取決于您的細胞特性。對于諸如HEK293，Hela，CHO，3T3，COS，HepG2，LNCaP，PC3，PC12，U2-OS，L929，MCF-7及Huh-7等常用細胞，一般的實驗步驟就能得到滿意的轉(zhuǎn)染結(jié)果。對于諸如MDCK，MDA-MB231，Caco-2，SaoS-2及HUVEC等難轉(zhuǎn)細胞，可以選用高級轉(zhuǎn)染步驟。針對您的細胞，若是您尚不明確選擇哪種轉(zhuǎn)染步驟，我們建議您可先嘗試一般步驟，如果您使用一般步驟得到的轉(zhuǎn)染效率低于5%，那么您的細胞比較難轉(zhuǎn)，您可嘗試高級轉(zhuǎn)染步驟。

Choose a more efficient protocol for your specific cell.

GenJet Ver. II, LipoD293 and PolyJet DNA in vitro transfection reagent all offer two protocols for transfecting mammalian cells--------a general protocol and an advanced protocol. The two protocol are written for transfecting different types of mammalian cells. The advanced protocol which involves a shaving cell technology is for very hard to transfect mammalian cells like MDCK, MDA-MB231, Caco-2, etc. The general protocol usually gives less than 5% efficiency while advanced protocol can get up to 60% efficiency on these problematic cells.

The principle to decide which one protocol is used to your cell is highly dependent upon the nature of your cell. For commonly used cells like HEK293, Hela, CHO, 3T3, COS, HepG2, LNCaP, PC3, PC12, U2-OS, L929, MCF-7 and Huh-7, be sure to use general protocol which usually gives very good transgene expression while the advanced protocol does not help. For very hard to transfect cells like MDCK, MDA-MB231, Caco-2, SaoS-2 and HUVEC, follow the advanced protocol by trypsinizing these adherent cells first followed by incubation of the cell pellet with transfection complex. If you do not know for your specific cells how to choose the more efficient protocol, we suggest you try general protocol first. If you get less than 5% efficiency with the general protocol, then your cell is hard to transfect in nature and you can consider trying advanced one.

如何優(yōu)化PolyJet轉(zhuǎn)染試劑

我們使用PolyJet DNA轉(zhuǎn)染試劑轉(zhuǎn)染表皮細胞及Raw267.4細胞非常有效，并且毒性很小。對于如何更好的優(yōu)化PolyJet轉(zhuǎn)染試劑，我樂意分享以下幾點：

1、DNA的質(zhì)與量。DNA的純度對于轉(zhuǎn)染實驗至關重要。由E Coli制備的DNA，A260/280必須達到1.80甚至更高。對于6孔板，通常每孔~1.0μg DNA足矣。其他規(guī)格的細胞培養(yǎng)皿，可以根據(jù)表面積適當調(diào)整DNA含量。

2、PolyJet/DNA的比率。對于表皮及Raw267.4細胞的轉(zhuǎn)染，我們將PolyJet/DNA的比率固定在3:1，并且得到了滿意的結(jié)果，沒有為尋找更合適的比率問題而煩惱。

3、稀釋。DNA及PolyJet的稀釋一定不要含有血清。我們使用的是不含血清的高糖DMEM培養(yǎng)基，效果很好。請不要選擇Opti-MEM，因為它可以影響轉(zhuǎn)染復合物的形成，因此，千萬不要使用Opti-MEM來稀釋質(zhì)粒及PolyJet。

4、轉(zhuǎn)染中可以含有血清。我們在含有血清/抗生素及不含二者的情況下分別進行可轉(zhuǎn)染實驗，沒有發(fā)現(xiàn)兩種情況下轉(zhuǎn)染效率有什么差別。因此，血清/抗生素對PolyJet轉(zhuǎn)染試劑沒有什么影響。

Some technical tips for optimizing PolyJet reagent.

PolyJet DNA Transfection Reagent is very efficient to transfect epithelial and Raw 267.4 cells in our hands without significant toxicity. Here I would like to share several technical points for better using PolyJet transfection reagent. 

1. DNA amount and quality. DNA purity is essential for high efficiency. DNA prepared from E Coli. must be 1.80 at A260/280 or higher. For 6-well plate, ~1.0 µg of DNA per well is usually good enough. For other cell culture format, just adjust DNA amount per culture dish's surface area.

2. Ratio of PolyJet/DNA. For epithelial and Raw 267.4 cells, we locked the ratio at 3:1 with excellent results and never bothered to find the optimal ratio. 

3. Diluent. Diluent used to dilute DNA and PolyJet reagent must be serum free. Serum-free DMEM medium with high glucose works for us. Opti-MEM is NOT a good choice because it may affect formation of transfection complex. So never use Opti-MEM as dilute. 

4. Transfection with serum. We performed transfection with or without serum/antibiotics and failed to find any difference in efficiency. Therefore, PolyJet reagent is NOT interfered by serum/antibiotics. 

如何優(yōu)化LipoD293轉(zhuǎn)染試劑

LipoD293DNA轉(zhuǎn)染試劑是脂質(zhì)體DNA轉(zhuǎn)運工具的升級版本。我們實驗室使用LipoD293DNA轉(zhuǎn)染試劑成功轉(zhuǎn)染了HepG2，LNCaP，CHO及HEK293細胞。

接下來，我們樂意就如何提高轉(zhuǎn)染效率，降低毒性等方面的小技巧同大家分享。

1、LipoD293/DNA的比率。盡管合適的比率由細胞類型決定，但是使用LipoD293/DNA，3:1的固定比率通常能得到很好的轉(zhuǎn)染效率。

2、每孔中DNA的含量。對于24孔板來說，我每孔使用的0.2到0.5µgDNA。太多的DNA（例如每孔1.0µg）沒有必要，并且會產(chǎn)生較高的毒性。其他規(guī)格的細胞培養(yǎng)器皿，可以根據(jù)表面積適當調(diào)整DNA含量。

3、DNA及LipoD293的稀釋。一定不要使用含有血清的培養(yǎng)基來稀釋二者。高糖的Plain DMEM培養(yǎng)基是不錯的選擇，但是，高糖并不是很重要。千萬不要使用Opti-MEM（invitrogen生產(chǎn)）！Opti-MEM可以影響轉(zhuǎn)染復合物的形成。我的同事沒有認識到該點的重要性，錯誤地使用了Opti-MEM，導致轉(zhuǎn)染效率低下。

4、血清/抗生素的存在與否對轉(zhuǎn)染沒有影響。目前，我們正在使用的哺乳動物細胞通常是用LipoD293進行轉(zhuǎn)染的，血清/抗清素對于轉(zhuǎn)染效率沒有影響。與其他依賴于脂質(zhì)體的轉(zhuǎn)染試劑不同，LipoD293不受血清/抗生素的影響，因此您不必擔憂轉(zhuǎn)染中含血清培養(yǎng)造成的高細胞死亡率問題。

5、轉(zhuǎn)染后更換培養(yǎng)基，對于我們正在研究的哺乳動物細胞，通常，我們是在轉(zhuǎn)染后24小時更換培養(yǎng)基，沒有必要在轉(zhuǎn)染后5小時更換培養(yǎng)基。

Some points which are important but ofetn neglected for optimizing LipoD293 reagent.

LipoD293 DNA Transfection Reagent is an enhanced version of liposome DNA delivery tool.  Our lab has been using LipoD293 DNA transfection reagent for transfecting HepG2, LNCaP, CHO and HEK293 cells with very good lucks. 
Now we like to share some technical points which I think are important for maximum efficiency and minimal toxicity and often ignored:

1. Ratio of LipoD293 reagent/DNA. While the optimal ratio is cell type dependent, the ratio of reagent/DNA locked at 3:1 often gives excellent efficiency. Our labs has been transfecting HepG2, LNCaP, CHO and HEK293 with LipoD293 reagent at 3:1 ratio with very good efficiency.

2. DNA amount per well. For 24-well plate, I used 0.2 to 0.5 µg of DNA per well. Too much DNA (eg, 1.0 µg per well) is unnecessary and might lead to high cytotoxicity. For other cell culture format, adjust DNA amount per culture dish's surface area.

3. Diluent for diluting DNA and LipoD293 reagent. Diluent must be serum free. Plain DMEM medium with high glucose is usually very good. High glucose seems not that important. Never use Opti-MEM (from Invitrogen)! This is very important 'cause my colleagues often failed to recognize importance of the diluent and misused Opti-MEM, leading to suboptimal efficiency. Per our initial test, Opti-MEM is able to disrupt formation of transfection complex.

4. Transfection with or without serum/antibiotics. For all mammalian cells we are currently working on, we always perform transfection with LipoD293 reagent in the presence of serum (10% FBS)/antibiotics without compromising any efficiency, thus greatly simplifying our transfection procedures. Unlike other liposome based reagent, LipoD293 reagent is usually NOT interfered by serum/antibiotics, so do not bother to perform transfection in serum-free medium which otherwise results in high cell death.

5. Medium change post transfection. We usually change medium 24 hours after transfection. Medium change 5 hours post transfection is not necessary for all the mammalian cell we are currency using.

轉(zhuǎn)染L929細胞的簡單步驟

L929是小鼠成纖維細胞瘤細胞株，經(jīng)常被用來檢測TNF-alpha及TNF-beta。對TNF的處理經(jīng)常會引發(fā)細胞凋亡及死亡，因此L929細胞經(jīng)常被用作免疫分析。但是，轉(zhuǎn)染L929細胞非常難，尤其使用基于脂質(zhì)體技術的轉(zhuǎn)染試劑，我們使用GenJet VerⅡ及PolyJet轉(zhuǎn)染L929細胞獲得了75%的轉(zhuǎn)染效率，簡單的實驗步驟如下。

1、轉(zhuǎn)染時確保L929細胞達到80%的融合度，細胞必須健康并且傳代不能超過9代。

2、對于6孔板，用不含血清的DMEM分別稀釋1.0µg，DNA及3.0µl，Genjet VerⅡ或PolyJet轉(zhuǎn)染試劑。將稀釋好的轉(zhuǎn)染試劑加入DNA中，室溫下放置15分鐘以形成轉(zhuǎn)染復合物，其它規(guī)格的細胞培養(yǎng)器皿，可以根據(jù)表面積適當調(diào)整DNA含量。

3、將轉(zhuǎn)染復合物直接加入L929細胞中；6孔板，每孔含1.0ml培養(yǎng)基，在血清/抗生素存在下,轉(zhuǎn)染進行。

4、轉(zhuǎn)染后的24~48小時，監(jiān)測轉(zhuǎn)染基因的表達情況。

Brief procedures for transfecting L929 cell.

L929 is a murine aneuploid fibrosarcoma cell line which is often used to assay TNF-alpha and TNF-beta. Treatment with TNF initiates apoptosis and subsequent cell death, therefore L929 cell is often used for immunological assays. However L929 cell is hard to transfect, especially resistant to liposome based transfection method. We have been using GenJet Ver. II and PolyJet to transfect L929 cell and got up to 75% efficiency. The brief procedures are described below for transfecting L929 cells with GenJet and PolyJet.

1. Grow L929 cell to ~80% confluency at the day of transfection. L929 cell must be healthy and less than 9 passages for maximum efficiency.

2. For 6-well plate, dilute 1.0 µg of DNA and 3.0 µl of GenJet Ver. II or PolyJet reagents per well with serum free DMEM respectively. Add diluted reagent to diluted DNA and let transfection complex formed at RT for 15 minutes.  For other format of cell culture formats, scale down or up per the surface area of culture dish.

3. Add transfection complex to L929 cell directly. Transfection is conducted in presence of serum/antibiotics with transfection volume of 1.0 ml per well of 6-well plate.

4. Check transgene expression 24~48 hours post transfection.

選擇GenJet^TM，LipoD293^TM & PolyJet^TMDNA轉(zhuǎn)染試劑稀釋溶液的小技巧

選擇何種稀釋液稀釋DNA及轉(zhuǎn)染試劑，對于制備有效的轉(zhuǎn)染復合物至關重要。除了溫度，孵育時間外，稀釋液的性質(zhì)對于制備高效的轉(zhuǎn)染復合物亦非常重要，同樣影響DNA轉(zhuǎn)染效率。根據(jù)我們的實驗數(shù)據(jù)，使用合適的稀釋液得到的轉(zhuǎn)染效率是使用錯誤稀釋液轉(zhuǎn)染效率的至少50倍。更為重要的是，實驗者總是忽視稀釋液的重要性，甚至一直未曾意識到正確的稀釋液對形成有效轉(zhuǎn)染復合物的重要性。

如下實驗技巧可以幫助您為GenJet^TM，LipoD293^TM&PolyJet^TMDNA轉(zhuǎn)染試劑選擇合適的稀釋液。

1、為了制備更有效的轉(zhuǎn)染復合物，稀釋液中千萬不要含有血清、蛋白。

2、盡量使用接近細胞培養(yǎng)基成分的稀釋液，例如：若是你使用DMEM（supplemented with serum and antibiotics）培養(yǎng)HEK293細胞，那么，最合適的稀釋液是不含血清、抗生素的DMEM：若是您使用RPM1-1640（supplemented with serum and antibiotics）培養(yǎng)Hela細胞，那么最合適的稀釋液是不含血清、抗生素的RPM1-1640。

3、請勿使用含有未知成分的培養(yǎng)基。例如，切勿使用Opti-MEM^TM稀釋GenJet™、PolyJet™及LipoD293™ DNA轉(zhuǎn)染試劑。根據(jù)我們的實驗數(shù)據(jù)，若是使用Opti-MEM稀釋轉(zhuǎn)染試劑及DNA，那么用于HEK293，Hela，CHO及NIH 3T3細胞的轉(zhuǎn)染效率至少減少50%。Opti-MEM^TM可能含有較少的血清，導致轉(zhuǎn)染效率的大幅降低。

4、不含血清的高糖DMEM是很好的選擇，可以與其他的生長培養(yǎng)基兼容，如果未能得到較高的轉(zhuǎn)染效率，您可能會考慮更換稀釋液，若是您不明確如何選擇合適的稀釋液，建議您初次可以嘗試不含血清的高糖DMEM，您應該會得到滿意的轉(zhuǎn)染效率。

Tricks in choosing diluent for GenJet™, LipoD293™ & PolyJet™ DNA transfection reagents.

For efficient formation of transfection complex, to choose an appropriate diluent which is used for diluting DNA transfection reagent and DNA is essential. In addition to temperature, incubation time, the nature of diluent is very important for complete formation of transfection complex, thus significantly affecting DNA transfection efficiency. Per our validation data, an appropriate diluent can be to 50 times more efficient than a "wrong" diluent. What's more important is researchers often neglect the importance of diluent and fail to recognize role of diluent in the process of transfection complex formation.

Follow the following technical tips to choose the proper diluent for GenJet™, PolyJet™ and LipoD293™ DNA transfection reagents:

1. For complete formation of transfection complex, the diluent must be serum-free and protein free. 

2. Try to use a diluent which is closest to the composition of cell culture medium. For instance, if you are using DMEM (supplemented with serum and antibiotics) to grow HEK293 cells, the best diluent is serum-free and antibiotics-free DMEM. If you are using RPMI-1640 Medium (supplemented with serum and antibiotics) to grow Hela cells, the best diluent is serum/antibiotic-free RPMI-1640 medium.

3. Never use a medium which contains unknown components. For example, never use Opti-MEM™ to dilute GenJet™, PolyJet™ and LipoD293™ DNA transfection reagents. Per our validation data, diluting our transfection reagents and DNA with Opti-MEM™ at least sacrificed 50% transfection efficiency on HEK293, Hela, CHO and NIH 3T3 cells. Also Opti-MEM™ may contains low level of serum which will greatly compromise transfection efficiency.

4. Basically serum-free DMEM with high glucose is pretty good choice. It is very easy accessible and compatible with other cell growth mediums. If you have trouble with lower transfection efficiency, you may think about changing diluent to serum-free DMEM with high glucose. If you do not know how to choose a proper diluent, you can try serum-free DMEM with high glucose first which usually gives you very good transfection efficiency.

報告基因檢測的精明之選—LipoD293DNA轉(zhuǎn)染試劑

使用lipoD293轉(zhuǎn)染試劑來轉(zhuǎn)染哺乳動物細胞（比如Hela 、PC3），以期檢測熒光酶報告基因，同其他轉(zhuǎn)染試劑相比較，您會得到更多的熒光酶讀數(shù)，更少的細胞死亡率。

使用unsupplemented RPM11640/DMEM替代Opti-MEM培養(yǎng)基來稀釋lipoD293及DNA（luciferase reporters），您會得到更高的熒光酶讀數(shù)（提高一倍），并且能節(jié)省更多的經(jīng)費。

如下是實驗過程中的幾個實驗要點：

1、使用相同類型的培養(yǎng)基（but unsupplemented）來稀釋lipoD293試劑及l(fā)uciferase reporterDNA（例如，若是細胞是用RPM11640-based培養(yǎng)基培養(yǎng)的，那么可選用unsupplemented RPM11640作為稀釋溶液）；

2、轉(zhuǎn)染前及轉(zhuǎn)染后，沒有必要更換培養(yǎng)基；

3、將稀釋的lipoD293試劑加入稀釋的DNA，混勻，在室溫下孵育15-20分鐘。

Smart choice for reporter assay with LipoD293 DNA transfection reagent.

Use lipoD293 DNA transfection reagent for transfection of multiple mammalian cell lines (such as Hela and PC3) with luciferase reporters, you will get much higher luciferase readings than other transfected reagents used on our lab for luciferase reading, but observe little cell death in transfected cells in our condition!

Use unsupplemented RPMI1640 / DMEM instead of Opti-MEM medium to dilute both lipoD293 and DNA (luciferase reporters), you will get further higher luciferase readings (one-fold increase) and save more money !

Several key points in the protocol:

1) Use the same type of culture medium (but unsupplemented) for diluting lipoD293 reagent and luciferase reporter DNA( for example, if the cells are cultured in RPMI1640-based medium, then use unsupplemented RPMI1640 as dilution medium);

3) No need to change medium either before or after transfection;

4) Add diluted lipoD293 reagent to diluted DNA and then incubate the mixture at room temperature for 15~20 minutes;

5) Make lysates for luciferase assay 16~48hrs post transfection

siRNA體外轉(zhuǎn)染

GenMute^TM siRNA體外轉(zhuǎn)染的優(yōu)化

GenMute^TM siRNA轉(zhuǎn)染試劑（Cat#SL100568）是市場上最有力的siRNA傳遞工具之一。最佳的siRNA濃度范圍是1.0nM到10nM，過多的siRNA可能導致沉默效果差的“洪水效應”。我們實驗室已經(jīng)使用GenMute^TM轉(zhuǎn)染試劑成功敲除了內(nèi)源表達的生長因子。以24孔板為例，如下步驟將對如何優(yōu)化GenMute^TM轉(zhuǎn)染試劑做一指導：至于其他規(guī)格器皿的培養(yǎng)的細胞，煩請參考GenMute^TM的實驗說明。

1、在轉(zhuǎn)染前的30~60分鐘，更換培養(yǎng)基，并在每孔中加入0.5ml的完全培養(yǎng)基（含血清和抗生素）。

2、將2.0pmol siRNA（5.0µM x 4.0µl）加入到盛有200µl GenMute轉(zhuǎn)染緩沖液（Cat#SL100572）的無菌管中進行稀釋，室溫下靜置5分鐘。

3、加入2.0µl GenMute轉(zhuǎn)染試劑，混勻，在室溫下放置15分鐘形成轉(zhuǎn)染復合物。請注意：室溫下，轉(zhuǎn)染復合物的靜置時間勿超過25分鐘。

4、取50µl、25µl、12.5µl轉(zhuǎn)染復合物分別加入細胞中（復孔），siRNA的終濃度各自達到10nM、5.0nM、1.25nM。

5、轉(zhuǎn)染24~48小時后，檢測4個不同siRNA濃度下的轉(zhuǎn)染效果，選擇出最佳的轉(zhuǎn)染條件。

Optimization of GenMute™ reagent for siRNA silencing.

GenMute™ siRNA transfection reagent (Cat # SL100568) is one of the most potent siRNA delivery tool in the market. The optimal siRNA concentrations range from 1.0 nM to 10 nM. Excessive siRNA may lead to "flooding effect" with sub-optimal silencing. Our lab has been using GenMute™ reagent to knock down endogenously expressed growth factors with very good luck.  The following procedures will guide to optimize GenMute™ reagent for best silencing in 24-well plate. For other cell culture formats, please refer to the protocol of GenMute™ reagent.

1). Change medium and add 0.5 ml of complete medium each well of 24-wel plate (with serum and antibiotics) 30 or 60 minutes before transfection.
2). Dilute 20 pmol siRNA (5.0 µM x 4.0 µl) into 200 µl of GenMute transfection buffer (Cat # SL100572) in a sterile tube and let's sit at RT for 5 minutes.
3). Add 2.0 µl GenMute reagent, briefly vortex and keep the transfection complex at RT for 15 minutes. Please note: never keep the transfection complex longer than 25 minutes at RT.
4). Add 50 µl transfection complex to your cells directly in duplicate (final 10 nM siRNA), 25 µl complex to another 2 wells (final 5.0 nM siRNA), 12.5 µl to 3rd 2 wells (final 2.5 nM siRNA) and 6.25 µl to 4th 2 wells (final 1.25 nM siRNA). 
5). Check silencing effect in the 4 different siRNA concentrations 24~48 hours post transfection and choose the best transfection conditions.

如何優(yōu)化GenMute^TM轉(zhuǎn)染試劑，提高siRNA/DNA共轉(zhuǎn)染效率

GenMute^TM siRNA轉(zhuǎn)染試劑（Cat#SL100568）是市場上最有力的siRNA傳遞工具之一。siRNA/DNA共轉(zhuǎn)染時，siRNA的最佳濃度范圍是0.5ηM到10ηM，過多的siRNA可能導致沉默效果差的“洪水效應”，切勿使用超過20ηM的siRNA。以24孔板為例，如下步驟將對如何優(yōu)化GenMute^TM轉(zhuǎn)染試劑做一指導；至于其他規(guī)格器皿的培養(yǎng)的細胞，煩請參考GenMute^TM的實驗說明。

1、在轉(zhuǎn)染前的30-60分鐘，更換培養(yǎng)基，并在每孔中加入0.5ml的完全培養(yǎng)基（含血清和抗生素）。

2、將0.5μg DNA各自加入到分別盛有50μl GenMute轉(zhuǎn)染緩沖液（Cat#SL100572）的4個無菌管中進行稀釋，接下來分別對4管中的siRNA進行系列稀釋·····將5.0pmol siRNA加入到第一管中，2.5 pmol siRNA加入到第二管中，1.25 pmol siRNA加入到第三管中，1.25 pmol siRNA加入到第四管中。室溫下靜置5分鐘。

3、取3.0μl GenMute^TM轉(zhuǎn)染試劑，分別加入到稀釋好的siRNA/DNA管中，混勻，室溫下靜置15分鐘以形成轉(zhuǎn)染復合物。請注意：室溫下，轉(zhuǎn)染復合物的靜置時間勿超過25分鐘。

4、將如上制備的轉(zhuǎn)染復合物分別加入24孔板中連續(xù)的4個孔中。

5、轉(zhuǎn)染24-48小時后，分別檢測4孔中的沉默效果并選擇出最佳的轉(zhuǎn)染條件。

Optimization of GenMute™ reagent for siRNA/DNA co-transfection.

GenMute™ siRNA transfection reagent (Cat # SL100568) is one of the most potent siRNA delivery tool in the market. The optimal siRNA concentrations for siRNA/DNA co-transfection range from 0.5 nM to 10 nM.  Excessive siRNA may lead to "flooding effect" which may comprise silencing effect, so never use siRNA higher than 20 nM. The following procedures will guide to optimize GenMute™ reagent for best siRNA/DNA co-transfection in 24-well plate. For other cell culture formats, please refer to the protocol of GenMute™ reagent.

1. Change medium and add 0.5 ml of complete medium to each well of 24-wel plate (with serum and antibiotics) 30 or 60 minutes before transfection.

2. Dilute 0.5 µg DNA to each sterile tube of total 4 tubes with 50 µl of GenMute transfection buffer (Cat # SL100572) followed by addition series diluted siRNA to each well of the total 4 tubes-------add 5.0 pmol siRNA to 1st tube, 2.5 pmols to 2nd tube, 1.25 pmols to 3rd tube and 1.25 pmols to 4th tube. Let's sit at RT for 5 minutes.

3. Add 3.0 µl GenMute™ reagent to diluted siRNA/DNA of each tube, briefly vortex and keep the transfection complex at RT for 15 minutes. Please note: never keep the transfection complex longer than 25 minutes at RT.

4. For 4 consecutive wells of 24-well plate, add the transfection complexes which are prepared with same concentration of DNA (0.5 µg per well) and different 4 concentrations of siRNA ranging from final 10 nM to 1.25 nM. 

5. Check silencing effect in the 4 wells 24~48 hours post transfection and choose the best transfection conditions.

如何有效地降低PepMute^TM，GenMute^TM and PepMute^TM Plus siRNA轉(zhuǎn)染試劑的毒性

PepMute^TM，GenMute^TM 及 PepMute^TM Plus轉(zhuǎn)染試劑，僅被發(fā)現(xiàn)在DMEM基礎細胞培養(yǎng)基中偶有毒性，使用其他的培養(yǎng)基（含10%FBS及抗生素）培養(yǎng)細胞，將基本減少轉(zhuǎn)染中的毒性。譬如，如果您發(fā)現(xiàn)有大量細胞死亡，您可能使用含10%FBS及抗生素的DMEM基礎培養(yǎng)基。要消除毒性，您可以選擇DMEM/F12或者RPMI 1640來替代DMEM作為基礎培養(yǎng)基。至于為什么，到目前為止，我們也是不得而知，但是，它卻有效地減少了毒性。試試吧！

How to eliminate toxicity of PepMute™, GenMute™ and PepMute™ Plus siRNA Reagents

PepMute™, GenMute™ and PepMute™ Plus siRNA transfection reagents are found to be sometimes toxic ONLY with DMEM base cell culture medium.  Using other cell culture mediums (supplemented with 10% FBS and antibiotics) to grow your cells will basically eliminate the transfection toxicity. For instance, if you find toxicity and high cell death, you are probably using DMEM base medium supplemented with 10% FBS and antibiotics to grow your cells.  To remove the transfection toxicity, you can change DMEM to DMEM/F12 or RPMI 1640 as base medium.   So far we have not figured out why, but it does minimize the toxicity.  Try it out!

SignaGen官方網(wǎng)站：http://

中國總經(jīng)銷 ：濟南美中清水灣生物科技有限公司

E-mail ： jnqsw@163.com

電話 ： 0531-85912162