诺唯赞试剂说明书 One Step Cloning kit

1、ClonExpress II One Step Cloning kitCatalog # C1121. IntroductionClonExpress One Step Cloning TechnologyClonExpress One Step Cloning Technology is a simple, fast and highly efficient cloning kit which is based on homologous recombination technology. It allows to directly clone any amplified product(s

2、) to any linearized vector, at any site. Firstly, the expression vector will be linearized at the cloning site of choice. A small sequence (15-20 bps) overlapped with the end of the cloning site will be added onto the insert through a PCR step. After the inserts and the linearized vector are mixed i

3、n the presence of Exnase for only 30 minutes, the cloning DNA products can be directly subjected to E.coli transformation with true positive rate over 95%.ClonExpress II One Step Cloning kit is a new version cloning kit based on ClonExpress One Step Cloning technology，supplied with Exnase II and rea

4、ction buffer. Compared to the previous version, Exnase II is added a unique recombinant enhancer which can significantly improve the efficiency of recombination (100 cfu/ ng Vector) even using low efficient competent cells (107 cfu/g). Additionally, Exnase II is also compatible with the endonuclease

5、 digesting reaction and PCR reaction assay. Digested products or PCR products can be directly applied in recombination reaction without purification, which significantly simplifies the experimental procedures. Product advantages-Easy and fast -Highly efficiency.-Applicable to multiple insert assembl

6、y at any cloning site of choice without restriction endonuclease -Sequentially assemble up to five inserts in a single reactionApplication -Fast assembly -High-through assembly-Seamless assembly-Site-specific mutagenesis2. Package InformationComponentsC112-01 (25 rxn)C112-02 (50 rxn)5 CE II Buffer10

7、0 l200 lExnase II50 l100 l500 bp control insert(25ng/l)5 l5 lpUC19 control vector, linearized (50 ng/l)5 l5 l3. StorageStore at -20C. 4. Protocols 4.1 Overview of the experimental process (Figure 1)Preparation for the linearized cloning vectors (see 4.2)；Design PCR primers of the insertions (see 4.3

8、)；PCR amplification of the insertions (see 4.4)；Recombination reaction (see 4.5)；Transformation and plating (see 4.6)；Selection of the positive colony (see 4.7). Figure1: Experimental procedures of the insert assembly with ClonExpressTM IIThe cloning vector must be linearized before applied to recom

9、bination reaction. The linearized vector can be obtained by digesting the circular vector with restriction endonuclease or by reverse PCR of the circular vector (Figure 1, upper left). The insertions are prepared by PCR amplification using primers with homologous recombination sequences added to the

10、ir 5 end (Figure 1, marked in blue and red). After PCR, identical sequences between different insertions and between insertions and the linearized cloning vectors are ready for homologous recombination (Figure 1, upper right). Set up the recombination reaction with linearized cloning vectors and the

11、 amplified insertions and react at 37Cfor 30 min to assemble insertion to cloning vector (Figure 1, middle). Transform the reaction products directly and hundreds of colonies will form on the plate for later positive selection (Figure 1, bottom). 4.2 Preparation for linearized cloning vectors Select

12、 appropriate cloning sites, and linearize the cloning vector. GC content of 20 bp regions at both ends of linearized cloning vector has great impacts on the recombination efficiency. The maximum recombination efficiency can be realized when the GC content of these regions is within 40%60%.Thus, its

13、better to avoid regions with sequence repeats and select regions containing even GC content.The cloning vectors can be linearized by restriction digesting with endonuclease or by reverse PCR amplification. Linearizing cloning vectors by restriction digestingDouble endonucleases digesting: Recommende

14、d due to the completeness of linearization and low transformation background (false positive clone).Single endonuclease digesting: Less extent of linearization compared to double digesting. Digestion time should be properly prolonged to lower the transformation background. Notice: There is no DNA li

15、gase in the recombination reaction and self-ligation of linearized vector will not occur. Therefore, dephosphorylation is unnecessary even if the linearized vectors are prepared by single endonuclease digesting. False positive clones (cloning vectors without insertions) appearing are mainly caused b

16、y vectors failed to be linearized. If a majority of clones are false positive, the linearized cloning vectors should be prepared anew. Exnase II is compatible with almost all digesting reaction. Therefore, after inactivating the endonuclease (see endonuclease manual, e.g. Hind III, incubate at 65C f

17、or 20 min to complete inactivate), the linearized cloning vector can be directly applied to recombination reaction. Preparing linearized cloning vectors via reverse PCR High-fidelity polymerase (Phanta Super-Fidelity DNA Polymerase, Vazyme, P501) is highly recommended for vector amplification to lim

18、it amplification errors. Try to use pre-linearized plasmids as templates to eliminate the transformation background induced by residual circular plasmids. Exnase II is compatible with conventional PCR reaction. Therefore, if the amplification template is pre-linearized plasmids and the amplification

19、 is highly specific, the PCR products can be applied directly to recombination reaction.The digesting products of the cloning vector or the PCR products may be relatively low in DNA purity and contain residual circular plasmids. Direct application of such products to recombination may lower the reco

20、mbination efficiency as well as the positive rate of cloning. Therefore, in case of large fragment (5kb) cloning, it is better to purify the linearized cloning vector and amplified insertions with gel recovery kit before recombination, so as to elevate DNA purity and get rid of parts of the residual

21、 circular vectors (its band position on the gel differs from that of the linearized cloning vector). Refer to Table 1 for the usages of the linearized cloning vectors prepared in different ways. Method to prepare linearized vectorTemplate typeFast experiment proceduresStandard experiment proceduresR

22、estriction digestingCircular plasmidDirect use after inactivating endonuclease by heatingGel recoveryReverse PCRAmplification is of high specificityCircular plasmidDirect use after DpnI digestion (degrade the PCR template)Gel recovery or gel recovery after DpnI digestionPre-linearized plasmid,genomi

23、c DNA, cDNADirect useGel recoveryShow apparent non-specific amplification Gel recovery Table 1: Usages of linearized cloning vectorsNotice：When applying digesting products or PCR products directly to recombination reaction, total volume used should be less than 4 l (1/5 of the recombination reaction

24、 volume). 4.3Design PCR primers of the insertionsThe principle for the design of ClonExpressTM II primers is: introduce homologous sequences (15 bp20 bp)into 5 end of primers, aiming to making the ends of amplified insertions and linearized cloning vector identical to the ends of their neighbours wh

25、ich is required for recombination reaction. Taking assembly of the insertion to pUC18 cloning vector as example, design the primers as Figure II:Figure 2: Design of recombinationNotice: If the primer length exceeds 40 bp, PAGE purification of synthetized primers is recommended, which will benefit th

26、e recombination efficiency. When calculating the Tm of primers, the homologous sequence of vector ends should be excluded and only gene specific amplification sequence should be counted.4.4 PCR amplification of insertionsInsertions can be amplified by any polymerase (Taq DNA polymerase or other high

27、-fidelity polymerases). It will not interfere with the recombination efficiency whether there is A-tail in the PCR products or not, which will be removed during recombination and missing in the final construct. To prevent mutations introduced during PCR, high-fidelity polymerases such as Phanta Supe

28、r-Fidelity DNA Polymerase (Vazyme, P501) is highly recommended.Take a small amount of products and run agrose electrophoresis after PCR to confirm the yields and specificity of amplification. Exnase MultiS is compatible with most PCR reactions. As a result, PCR products can be directly applied to re

29、combination reaction without further purification if the PCR templates are not circular plasmids which share the same antibiotic resistance with the cloning vector.Direct application of PCR products to recombination reaction may sometimes decrease the efficiency and positive rate of cloning due to i

30、ts low purity. Thus, in case of long insertions (5 kb), we recommend a pre-purification step of PCR products with commercial gel recovery kits to elevate their purity. Amplified insertions can be used according to Table 2.PCR AmplificationTemplate typeFast experiment proceduresStandard experiment pr

31、oceduresAmplification is of high specificityCircular plasmids sharing the same antibiotic resistance with the cloning vectorDirect use after DpnI digestion*Direct gel recovery or gel recovery after DpnI digestionPre-linearized plasmid, genomic DNA, cDNADirectly useGel recoveryShow apparent non-speci

32、fic amplification Gel recoveryTable 2: Usages of amplified insertionsNotice: When applied to recombination reactions without gel recovery, the volume of PCR products should be less than 4 l (1/5 of the total volume of the recombination reaction)*If linearized cloning vectors are prepared by digestin

33、g circular plasmids, PCR products after DpnI digestion should be incubated at 85C for 20 min to deactivate DpnIso as to prevent linearized cloning vectors from degradation.4.5 Recombination reactionSet up the following reaction on ice. Spin briefly to bring the sample to the bottom before reacting.d

34、dH2OUp to 20 l5CE II Buffer4 lLinearized cloning vector50200 ngPCR products of insertions50200 ngExnase II2 lThe recommended amount of vector for recombination reaction is 0.03 pmol. The recommended amount of insertion for recombination reaction is 0.06 pmol. Their corresponding mass can be roughly

35、calculated according the following formula:The mass of vector required = 0.02number of base pair ng (0.03 pmol)The mass of fragment required = 0.04number of base pair ng (0.06 pmol)For example, when cloning the insertion of 2 kb to a 5 kb vector, their corresponding DNA mass needed is as follows:Lin

36、earized cloning vector: 0.025000 = 100 ng2 kb insertion: 0.042000 = 80 ngNotice: The mass of linearized cloning vector used should be between 50200 ng. Use 50 or 200 ng if the calculated mass is out of range.The mass of insertions should be over 10 ng. Use 10 ng if the calculated mass is less.When a

37、pplied to recombination reaction without gel recovery, the total volume of unpurified DNA used should be less than 1/5 of that of recombination reaction, which is 4 l.ClonExpress II One Step Cloning Kit provides 5 l of pUC19 control vector (linearized, 50 ng/l) and 5 l of control insert (0.5 kb, 25

38、ng/ l). Use 1 l of each in one reaction of positive control.After finishing setting up, gently pipette up and down several times with a pipettor to mix thoroughly and try to avoid the formation of bubbles. DO NOT VOTEX. Incubate the reaction at 37Cfor 30 min and immediately place it on ice for 5 min

39、. Recombination product is now ready for transformation, or otherwise it can be stored at -20C before transformation.Notice: PCR thermo cyclers or water bath are recommended for reaction. The recombination efficiency can reach its peak at 30 min. Longer or shorter reaction time will influence the cl

40、oning efficiency.4.6 Transformation and platingAdd the entire recombination products to 200 l of competent cells; flip the tube several times to mix it thoroughly and place the tube on ice for 30 min. Heat-shock the tube for 4590 sec at 42C and then place the tube on ice for 2 min. Add 900 l of SOC

41、or LB medium to competent cells and leave the tube in 37C water bath for 10 min to let the competent cells fully recovered. Then, shake the tube at 37C for 45 min to culture the bacteria. Take 100 l of culture and plate evenly on agar plate which contains appropriate selection antibiotic. Place the

42、plate at 37C overnight to culture.Notice: Competent cells with transformation efficiency over 108 cfu/g are highly recommended. If not, it is necessary to centrifuge the culture at 5000 rpm for 3 min to collect the bacteria before plating. Then, resuspend the pellet with 100 l of LB medium and plate

43、 all.4.7 Selection of positive colonyColony PCR is the most convenient selection method. Pick a single colony with tips to 2050 l of LB medium, mix thoroughly and take 1 l as PCR template. To avoid false positive PCR, we recommend at least one sequencing primer of the cloning vector should be used.

44、Inoculate the remaining medium of positive clones into fresh LB medium and culture overnight. Then, extract the plasmids for subsequent authentication. Examples for reference (Control reaction)Purpose: Insert the 0.5 kb fragment to pUC19 vector at EcoRI and HindIII recognition sites. Design PCR prim

45、ers for insertions Design the primers according to Figure 2 (Figure 4):Figure 4: Design of the forward primer for 0.5 kb insertion and the reverse primer for 2 kb insertionDesign the reverse primer of 0.5 kb insertion and the forward primer of 1 kb insertion according to Figure 3 (Figure 5):Figure 5

46、: Design of the reverse primer for 0.5 kb insertion and the forward primer for 1 kb insertionDesign the reverse primer of 1 kb insertion and the forward primer of 2 kb insertion according to Figure 3 (Figure 6):Figure 6: Design of the reverse primer for 1 kb insertion and the forward primer for 2 kb

47、 insertionLinearization of the cloning vectors Digest 2 g circular pUC19 vector in 20 l reaction system at 37C for 2 hours. The amount of endonucleases is 1l respectively for EcoRI and HindIII. Heat the reaction at 65C for 20 min to deactivate the endonucleases. PCR amplification of the insertions A

48、mplify the three insertions with Phanta Super-Fidelity DNA Polymerase (Vazyme, P501) using genomic DNA as templates. Take a small amount of the amplification products and run agarose electrophoresis. The amplification was of high specificity. Determination of DNA concentrationTake 1 lof the lineariz

49、ed cloning vector and amplified insertions and dilute with ddH2O to a given fold (indicated in Figure 7). After electrophoresis, compare the intensity of their bands with DNA Marker to determine the approximate concentration (most DNA maker gives determined mass for each band when a certainvolume is

50、 loaded, Figure 7). It can be seen that the DNA concentration of linearized pUC19 is approximately 100 ng/l; the DNA concentrations of the amplified insertionsareall around 400 ng/l. Figure 7: Determination of DNA concentration via agarose electrophoresisM: DNA Marker III. When 5 l is loaded，DNA mas

51、s of each band is 50 ng except 1.2 kb which is 100 ng. It can be seen that after linearized pUC19 is diluted by one fold and 1 l is loaded its band intensity is similar to that of 3.0 kb band of DNA Marker III (indicated by the orange box). Therefore, the concentration of linearized pUC19 is estimat

52、ed to be 50 ng2 = 100 ng/l; the intensity of 0.5 kb insertion which is diluted by seven fold and 1 l of which is loaded is similar to that of 0.5 kb band of DNA Marker III (indicated by the purple box). Therefore, the concentration of 0.5 kb insertion is estimated to be 50 ng 8 = 400 ng/l; the inten

53、sity of 1 kb insertion which is diluted by three fold and 1 l of which is loaded is similar to that of 1.2 kb band of DNA Marker III (indicated by the gray box). Therefore, the concentration of 1 kb insertion is estimated to be 100 ng 4 = 400 ng/l; the intensity of 2 kb insertion which is diluted by

54、 seven fold and 1 l of which is loaded is similar to that of 2.0 kb band of DNA Marker III (indicated by blue box). Therefore, the concentration of 2 kb insertion is estimated to be 50 ng8 = 400 ng/l.Setting-up of the reactionCalculate the DNA amount required for recombination according to section 4

55、.5 Recombination reaction.Linearized pUC19 vector: 0.022700 (2700 bp) 50 ng0.5 kb insertion: 0.04500 (500 bp) 20 ngDilute the linearized pUC19 vector with ddH2O to 50 ng/l, 0.5 kb insertion to 20 ng/l. Set up the following reaction on ice:Recombination reactionNegative control*ddH2O12 l18 lLinearize

56、d pUC19 vector ( 50 ng/l)1 l1 l0.5 kb insertion ( 20 ng/l)1 l1 l5CE II Buffer4 l0 lExnase II2 l0 lTotal20 l20 l*Negative control is recommended to determine the amount of residual circular plasmids with the same antibiotic resistance to the cloning vector in the linearized cloning vector and the amp

57、lified insertions.Gently pipette the reaction up and down for several times with a pipettor to mix thoroughly. Incubate the reaction at 37C for 30 min and immediately place it on ice for 5 min. Transformation and platingRefer to section 4.6 Transformation and plating. The transformation efficiency o

58、f competent cells used was 107 cfu/g. After culture, bacteria were collected by centrifuging at 5000 rpm for 3 min and the pellet was resuspended with 100 l of LB medium and plated all.Selection of positive colonyAfter overnight culture, hundreds of colonies formed on the plate transformed with reco

59、mbination reaction whereas there were fewer on the plate transformed with negative control (Figure 8). Colony PCR was carried out for the 14 randomly picked colonies by using vector sequencing primers M13F and RV-M (Taq Plus Master Mix, Vazyme, P212). Positive colony should give a band of 3.5 kb PCR

60、 product. It can be seen that 13 out of the 14 colonies were positive (except 3#, Figure 9), which were further confirmed by double digesting by EcoRI and BamHI (Figure 10).Figure 8: Agar plate after overnight cultureFigure 9: Colony PCR of randomly picked 14 coloniesM: Marker III; 1-14, 14 colonies