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View additional product information for Gateway™ pDONR™221 Vector - FAQs (12536017)
57 product FAQs found
•检查反应产物是否被转化进了包含F'episome和ccdA基因的 E. coli 菌株中（请使用不包含F'episome的 E. coli 菌株，例如DH10B, TOP10）。
•检查ccdB基因是否有缺失（全部或部分）（在含有50-100 mg/mL氨苄和15-30 µg/mL氯霉素的培液中扩增）。
•检查是否有其它抗性菌株污染。
•检查反应中是否使用了合适用量的DNA。
通常，当BP重组后产生大的和小的菌落时，我们建议选择几个大的和小的菌落类型，通过分析限制性内切酶消化结果、基因特异性引物的PCR或测序进行筛选，以确定你拥有什么，并决定最佳的前进方向。如果您分析的菌落中有一个包含您想要的入门克隆，那么您可以将该入门克隆进行LR反应，以生成您想要的表达克隆。
这个问题有几个可能的原因：
•质粒由于太大或有毒性而在培养过程中丢失；为了改善使用棘手的插入片段进行BP反应的结果，下次您可以尝试在30摄氏度下孵育转化后平板和/或使用Stbl2 E. coli来稳定质粒。我们也推荐在做感兴趣的BP克隆反应的同时用pEXP7-Tet质粒进行BP反应阳性对照；对照反应的结果会让你知道大的和小的菌落是否是你感兴趣的插入片段特异的表型。
•ccdB基因发生缺失（部分或全部）或点突变；BP反应阴性对照(不添加BP克隆酶)不应产生任何菌落–如果无BP克隆酶的阴性对照出现菌落，则ccdB/氯霉素表达框受损，我们建议使用新的pDONR载体。
•污染或抗生素过期导致抗生素筛选平板上出现背景菌落 - 如果你做一个无质粒添加的转化阴性对照平板，那么这个平板上不应该产生任何菌落。转化阴性对照平板上的菌落表明有污染或需要制作新的平板了。
•使用pUC19进行转化以检查感受态细胞是否正常。
•提高铺板使用的菌量。
•延长孵育时间至最长18小时。
•确保转化前使用蛋白酶K处理反应产物。
•检查是否使用了正确的抗生素进行筛选。
•检查att位点序列是否正确。
•检查是否使用了正确的Clonase酶以及它是否功能正常。
•检查是否在反应中使用了推荐量的DNA。
•检查引物设计并试一试使用凝胶/PEG纯化attB-PCR产物。
•如果attB-PCR产物或线性attB表达克隆太大（>5 kb），请将BP反应物孵育过夜。
您可以继续，但重组效率将降低大约10倍。
要实现最高效的BP反应，最好要使attB位点的摩尔数不要超过attP位点的。标准的BP反应（20 µL）使用300 ng (不超过500 ng)的pDONR载体和30-300 ng两侧带attB位点的PCR产物或表达克隆在25摄氏度孵育1小时。在反应中使用过多的供体载体将抑制BP反应并且会导致完整的供体载体和入门载体共转化。这将降低平板上的克隆数，因为ccdB基因的存在会杀死转化后的E. coli。将孵育时间延长至24小时可以将更大比例的起始attB-DNA转化为产物。对于> 4 kb的PCR产物，每fmol PCR DNA获得的克隆数随着片段大小的增加而下降。因此，对于较大的PCR产物，我们推荐将每20 µL反应的PCR产物用量提高到至少100 fmol，并使用超过1小时的孵育时间（例如6小时或孵育过夜到24小时）。我们自己克隆过的最大PCR片段是10.1 kb。将孵育时间延长至4-6小时通常会将克隆产出提高2-3倍，而孵育时间延长至16-24小时会将克隆产出延长5-10倍。
pDONR221和pDONR/Zeo载体的骨架完全相同，除了抗生素抗性标记不同。pDONR221带有卡那霉素抗性标记，而pDONR/Zeo带有Zeocin抗性标记。
可以使用pDONR201和pDONR207，但是尽管pDONR201和pDONR207都含有pUC复制起始点，它们的复制效率却低于我们提供的pDONR221和pDONR/Zeo供体载体，这导致质粒的产量较低。使用pDONR221时，质粒的产量范围是每毫升菌液0.5 - 1.0 µg DNA。
理论上，pDONR载体在BP反应时对插入片段没有大小的限制。我们自己测试过的最大片段是12 kb。TOPO载体对插入片段大小更敏感一些，要获得较高的克隆效率其插入片段长度的上限是3-5 kb。
在得到attB-PCR产物之后，我们建议对产物进行纯化以去除PCR缓冲液，残留的dNTP，attB引物，以及attB引物二聚体。引物和引物二聚体在BP反应中会高效的与供体载体重组，因而会增加转化E. coli时的背景，而残留的PCR缓冲液可能会抑制BP反应。使用酚/氯仿抽提，加醋酸铵和乙醇或异丙醇沉淀的标准PCR产物纯化方案不适合对attB-PCR产物进行纯化，因为这些实验方案通常仅能去除小于100 bp的杂质，而在去除较大的引物二聚体时效果不佳。我们推荐一种PEG纯化方案（请参见使用Clonase II的Gateway技术手册第17页）。如果使用上述实验方案您的attB-PCR产物仍然不够纯，您可以进一步对其进行凝胶纯化。我们推荐使用Purelink Quick 凝胶纯化试剂盒。
请检查您所用的菌株的基因型。我们的Gateway目的载体通常含有一个ccdB基因元件，该元件如果不被破坏，则E. Coli生长将受到抑制。因此，未进行克隆的载体应该在ccdB survival菌株如我们的ccdB Survival 2 T1R感受态细胞中扩增。
目的基因必须两端带有合适的att位点，或者是入门克隆中的attL (100 bp)位点，或者是PCR产物中的 attB (25 bp)位点。对于入门克隆而言，所有位于attL位点之间的部分都将被转移到含有attR位点的Gateway目的载体中，而两端带有attB位点的PCR产物需被转移到一个含有attP位点的供体载体，例如pDONR221。
翻译起始位点的位置，终止子，或者用于表达的融合标签必须在最开始的克隆设计中考虑到。例如，如果您的目的载体包含一个N末端标记而非C末端标记，则该载体应当已经带有合适的翻译起始位点，但是终止子应当被包含在插入片段当中。
小抽（碱裂解）纯化的DNA即适用在Gateway克隆反应中。重要的一点是要将RNA污染去除干净以便得到精确的定量。推荐使用通过我们的S.N.A.P. 核酸纯化试剂盒，ChargeSwitch试剂盒，或PureLink试剂盒纯化的质粒DNA。
理论上没有片段大小限制。长度在100 bp到11 kb之间的PCR产物可以被直接克隆到pDONR Gateway载体中。其它DNA片段如带有att位点的150 kb DNA片段可以成功和一个Gateway兼容载体发生重组。对于大的插入片段，推荐进行过夜孵育反应。
– Check whether the reaction was transformed into an E.coli strain containing the F' episome and the ccdA gene – use an E.coli strain that does not contain the F' episome, e.g. DH10B, TOP10.
– Deletion (full or partial) of the ccdB gene – propagate in media with 50-100 mg/mL ampicillin and 15-30 µg/mL chloramphenicol.
– Contamination from another resistant strain.
– Check whether proper amount of DNA was used in the reaction.
Typically when both large and small colonies are produced following BP recombination, we recommend screening several of both of the small and large colony types by analytical restriction digest, PCR with gene specific primers, or by sequencing to determine what you have and decide on the best course forward. If one of the colonies you analyze contains your desired entry clone, then you may proceed with this entry clone to an LR reaction to produce your desired expression clone.
There are several possible causes of this issue:
– Plasmid was lost during culture due to large size or toxicity – To improve results next time you perform a BP reaction with a tricky insert, you may try incubating your transformation plate at 30 degrees C and/or use Stbl2 E.coli to stabilize the plasmid. We also recommend performing the BP reaction positive control pEXP7-Tet alongside your BP cloning reaction of interest; the results of that control reaction will let you know whether the large and small colony phenotype is specific to your insert of interest.
– Deletions (full or partial) or point mutations in the ccdB gene – A negative BP reaction control (with no BP clonase added) should not produce any colonies – If a no BP clonase negative control produces colonies, then the ccdB/chlorophenicol cassette is compromised and we recommend to obtain a new pDONR vector.br/>
– Background on antibiotic selection plate due to contamination or expired antibiotic – If you run a no plasmid added transformation negative control plate, then this plate should not produce any colonies. Colonies on transformation negative control plate suggests contamination or need to make fresh plates.
– Check the competent cells with pUC19 transformation.
– Increase the amount plated.
– Increase the incubation time up to 18 hours.
– Make sure to treat reactions with proteinase K before transformation.
– Check whether the correct antibiotic was used for selection.
– Check whether the att site sequences are correct.
– Check whether the correct Clonase enzyme was used and whether it was functional.
– Check whether the recommended amount of DNA was used in the reaction.
– Check primer design and try gel/PEG purifying the attB-PCR product.
– If the attB-PCR product or linear attB Expression clone is too long (>5 kb), incubate the BP reaction overnight.
You may continue, but the recombination efficiency will drop by approximately 10 fold.
For the most efficient BP reaction, it is best to not have attB sites in molar excess of attP sites. The standard BP Reaction (20 µL) uses 300 ng (no more than 500 ng) of pDONR vector and 30-300 ng attB-flanked PCR product or Expression Clone for 1 hour at 25 degrees C. Using too much of the Donor vector in the reaction tube will inhibit the BP reaction and also result in intact donor vector being co-transformed with the Entry Clones. This will reduce the number of colonies on the plate by killing the transformed E. coli due to the presence of the ccdB gene. Longer incubation times of up to 24 hours can be used to convert a higher percentage of starting attB-DNA to product. For PCR products > 4 kb, the number of colonies obtained per fmol of PCR DNA added decreases with increasing size. Thus, for larger PCR products, it is recommended to increase the amount of DNA to at least 100 fmol of PCR product per 20 µL reaction, and using incubations longer than one hour (e.g., 6 hours or overnight to 24 hours). The largest PCR-amplified DNA cloned in-house was 10.1 kb. Increasing the incubation to 4-6 hours will typically increase colony output 2-3 fold and 16-24 hours will typically increase colony output 5-10 fold.
The backbones of the pDONR221 and pDONR/Zeo vectors are exactly the same except for the antibiotic resistance marker. pDONR221 has the kanamycin antibiotic resistance marker, while pDONR/Zeo has the zeocin resistance marker.
pDONR201 and pDONR207 can be used, but they replicate less efficiently than the Donor vectors we offer, pDONR221 and pDONR/Zeo, even though both pDONR201 and pDONR207 contain the pUC ori. This results in lower plasmid yields. With pDONR221, plasmid yields are in the range of 0.5 - 1.0 µg of DNA per mL of culture.
There is no theoretical limit to insert size for a BP reaction with a pDONR vector. Maximum size tested in-house is 12 kb. TOPO vectors are more sensitive to insert size and 3-5 kb is the upper limit for decent cloning efficiency.
After generating your attB-PCR product, we recommend purifying it to remove PCR buffer, unincorporated dNTPs, attB primers, and any attB primer-dimers. Primers and primer-dimers can recombine efficiently with the Donor vector in the BP reaction and may increase background after transformation into E. coli, whereas leftover PCR buffer may inhibit the BP reaction. Standard PCR product purification protocols using phenol/chloroform extraction followed by ammonium acetate and ethanol or isopropanol precipitation are not recommended for purification of the attB-PCR product as these protocols generally have exclusion limits of less than 100 bp and do not efficiently remove large primer-dimer products. We recommend a PEG purification protocol (see page 17 of the Gateway Technology with Clonase II manual). If you use the above protocol and your attB-PCR product is still not suitably purified, you may further gel-purify the product. We recommend using the PureLink Quick Gel Extraction kit.
Check the genotype of the cell strain you are using. Our Gateway destination vectors typically contain a ccdB cassette, which, if uninterrupted, will inhibit E. coli growth. Therefore, un-cloned vectors should be propagated in a ccdB survival cell strain, such as our ccdB Survival 2 T1R competent cells.
LR Clonase II Plus contains an optimized formulation of recombination enzymes for use in MultiSite Gateway LR reactions. LR Clonase and LR Clonase II enzyme mixes are not recommended for MultiSite Gateway LR recombination reactions, but LR Clonase II Plus is compatible with both multi-site and single-site LR recombination reactions.
When the LR reaction is complete, the reaction is stopped with Proteinase K and transformed into E. coli resulting in an expression clone containing a gene of interest. A typical LR reaction followed by Proteinase K treatment yields about 35,000 to 150,000 colonies per 20ul reaction. Without the Proteinase K treatment, up to a 10 fold reduction in the number of colonies can be observed. Despite this reduction, there are often still enough colonies containing the gene of interest to proceed with your experiment, so the Proteinase K step can be left out after the LR reaction is complete if necessary.
In most cases, there will not be enough pENTR vector DNA present to go directly from TOPO cloning into an LR reaction. You need between 100-300 ng of pENTR vector for an efficient LR reaction, and miniprep of a colony from the TOPO transformation is necessary to obtain that much DNA. However, if you want to try it, here are some recommendations for attempting to go straight into LR reactions from the TOPO reaction using pENTR/D, or SD TOPO, or pCR8/GW/TOPO vectors:
1. Heat inactivate the topoisomerase after the TOPO cloning reaction by incubating the reaction at 85 degrees C for 15 minutes.
2. Use the entire reaction (6 µL) in the LR clonase reaction. No purification steps are necessary.
3. Divide the completed LR reaction into 4 tubes and carry out transformations with each tube. You cannot transform entire 20 µL reaction in one transformation, and we have not tried ethanol precipitation and then a single transformation.
When attempting this protocol, we observed very low efficiencies (~10 colonies/plate). So just be aware that while technically possible, going directly into an LR reaction from a TOPO reaction is very inefficient and will result in a very low colony number, if any at all.
To have an N-terminal tag, the gene of interest must be in the correct reading frame when using non-TOPO adapted Gateway entry vectors. All TOPO adapted Gateway Entry vectors will automatically put the insert into the correct reading frame, and to add the N-terminal tag you simply recombine with a destination vector that has N-terminal tag.
To attach a C-terminal tag to your gene of interest, the insert must lack its stop codon, and be in the correct reading frame for compatibility with our C-terminal tagged destination vectors. Again, TOPO adapted Gateway Entry vectors will automatically put the insert into the correct reading frame. If you do not want the C-terminal tag to be expressed, simply include a stop codon at the end of the insert that is in frame with the initial ATG.
Generally, you need to choose a destination vector before you design and clone your insert into the Entry vector. This will determine whether you need to include an initiating ATG or stop codon with your insert.
No, not directly. The attB-PCR product must first be cloned, via a BP Clonase reaction, into a pDONR vector which creates an "Entry Clone" with attL sites. This clone can then be recombined, via an LR Clonase reaction, with a Destination vector containing attR sites. However, It is possible to perform both of these reactions in one step using the "One-Tube Protocol" described in the manual entitled "Gateway Technology with Clonase II".
Yes, this can be done using the Multisite Gateway Technology. MultiSite Gateway Pro Technology enables you to efficiently and conveniently assemble multiple DNA fragments - including genes of interest, promoters, and IRES sequences - in the desired order and orientation into a Gateway Expression vector. Using specifically designed att sites for recombinational cloning, you can clone two, three, or four DNA fragments into any Gateway Destination vector containing attR1 and attR2 sites. The resulting expression clone is ready for downstream expression and analysis applications.
For the BP reaction, approximately 5-10% of the starting material is converted into product. For the LR reaction, approximately 30% of the starting material is converted into product.
The core region of the att sites contains the recognition sequence for the restriction enzyme BsrGI. Provided there are no BsrGI sites in the insert, this enzyme can be used to excise the full gene from most Gateway plasmids. The BsrGI recognition site is 5'-TGTACA and is found in both att sites flanking the insertion site.
If a different restriction site is desired, the appropriate sequence should be incorporated into your insert by PCR.
We do have an alternative method called the "attB Adapter PCR" Protocol in which you make your gene specific primer with only 12 additional attB bases and use attB universal adapter primers. This protocol allows for shorter primers to amplify attB-PCR products by utilizing four primers instead of the usual two in a PCR reaction. You can find the sequence of these primers in the protocol on page 45 of the "Gateway Technology with Clonase II" manual.
There is a protocol in which all 4 primers mentioned above are in a single PCR reaction. You can find this protocol at in the following article: Quest vol. 1, Issue 2, 2004. https://www.thermofisher.com/us/en/home/references/newsletters-and-journals/quest-archive.reg.in.html. The best ratio of the first gene-specific and the second attB primers was 1:10.
We do not offer pre-made primers, but we can recommend the following sequences that can be ordered as custom primers for sequencing of pDONR201:
Forward primer, proximal to attL1: 5'- TCGCGTTAACGCTAGCATGGATCTC
Reverse primer, proximal to attL2: 5'-GTAACATCAGAGATTTTGAGACAC
1. Yeast two-hybrid protein-protein interaction studies Walhout AJ, Sordella R, Lu X, Hartley JL, Temple GF, Brasch MA, Thierry-Mieg N, Vidal M.
2. Protein Interaction Mapping in C. elegans Using Proteins Involved in Vulval Development. Science Jan 7th 2000; 287(5450), 116-122 Davy, A. et al.
3. A protein-protein interaction map of the Caenorhabditis elegans 26S proteosome. EMBO Reports (2001) 2 (9), p. 821-828. Walhout, A.J.M. and Vidal, M. (2001).
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5. Large Scale Analysis of Protein Complexes Gavin, AC et al. Functional Organization of the Yeast Proteome by Systematic Analysis of Protein Complexes. Nature Jan 10th 2002, 415, p. 141-147.
6. Systematic subcellular localisation of proteins Simpson, J.C., Wellenreuther, R., Poustka, A., Pepperkok, R. and Wiemann, S.
7. Systematic subcellular localization of novel proteins identified by large-scale cDNA sequencing. EMBO Reports (2000) 1(3), pp. 287-292.
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15. Reviewed in NATURE: Free Access to cDNA provides impetus to gene function work. 15 march 2001, p. 289. Generating directional cDNA libraries using recombination
16. Osamu Ohara and Gary F. Temple. Directional cDNA library construction assisted by the in vitro recombination reaction. Nucleic Acids Research 2001, Vol. 29, no. 4. RNA interference (RNAi)
17. Varsha Wesley, S. et al. Construct design for efficient, effective and highthroughput gene silencing in plants. The Plant Journal 27(6), 581-590 (2001). Generation of retroviral constructs
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21. Kneidinger, B. et al. Identification of two GDP-6-deoxy-D-lyxo-4-hexulose reductase synthesizing GDP-D-rhamnose in Aneurinibacillus thermoaerophilus L420-91T*. JBC 276(8) (2001).
The attP1 sequence (pDONR) is:
AATAATGATT TTATTTTGAC TGATAGTGAC CTGTTCGTTG CAACAAATTG ATGAGCAATGCTTTTTTAT AATGCCAACT TTGTACAAAA AAGC[TGAACG AGAAACGTAA AATGATATAA ATATCAATAT ATTAAATTAG ATTTTGCATA AAAAACAGACTA CATAATACTG TAAAACACAA CATATCCAGT CACTATGAAT CAACTACTTA GATGGTATTA GTGACCTGTA]
The region within brackets is where the site is "cut" and replaced by the attB1-fragment sequence to make an attL1 site. The sequence GTACAAA is the overlap sequence present in all att1 sites and is always "cut" right before the first G.
The overlap sequence in attP2 sites is CTTGTAC and cut before C. This is attP2:
ACAGGTCACT AATACCATCT AAGTAGTTGA TTCATAGTGA CTGGATATGT TGTGTTTTAC AGTATTATGT AGTCTGTTTT TTATGCAAAA TCTAATTTAA TATATTGATA TTTATATCAT TTTACGTTTC TCGTTCAGCT TTCTTGTACA AAGTTGGCAT TATAAGAAAG CATTGCTTAT AATTTGTTG CAACGAACAG GTCACTATCA GTCAAAATAA AATCATTATT
So, attL1 (Entry Clone) should be:
A ATAATGATTT TATTTTGACT GATAGTGACC TGTTCGTTGC AACAAATTGA TGAGCAATGC TTTTTTATAA TGCCAACT TT G TAC AAA AAA GC[A GGC T]NN NNN
attL2 (Entry Clone) should be:
NNN N[AC C]CA GCT TT CTTGTACA AAGTTGGCAT TATAAGAAAG CATTGCTTAT CAATTTGTTG CAACGAACAG GTCACTATCA GTCAAAATAA AATCATTATT
The sequence in brackets comes from attB, and N is your gene-specific sequence.
Note: When creating an Entry Clone through the BP reaction and a PCR product, the vector backbone is not the same as Gateway Entry vectors. The backbone in the case of PCR BP cloning is pDONR201.
There is no size restriction on the PCR fragments if they are cloned into a pDONR vector. The upper limit for efficient cloning into a TOPO adapted Gateway Entry vector is approximately 5 kb. A Gateway recombination reaction can occur between DNA fragments that are as large as 150 kb.
Destination vectors that contain N-terminal fusion partners will express proteins that contain amino acids contributed from the attB1 site, which is 25 bases long. This means that in addition to any tag (6x His and/or antibody epitope tag), the N-terminus of an expressed protein will contain an additional 9 amino acids from the attB1 sequence - the typical amino acid sequence is Thr-Ser-Leu-Tyr-Lys-Lys-Ala-Gly-nnn, where nnn will depend on the codon sequence of the insert.
Effects on protein function: A researcher (Simpson et al. EMBO Reports 11(31):287-292, 2000) demonstrated that GFP fusions (N- terminal and C-terminal) localized to the proper intracellular compartment. The expression constructs were generated using Gateway cloning, so the recombinant protein contained the attB1 or attB2 amino acid sequence. The localization function of the cloned recombinant proteins was preserved.
Effects on expression: We have seen no effect of the attB sites on expression levels in E. coli, insect and mammalian cells. The gus gene was cloned into bacterial expression vectors (for native and N-terminal fusion protein expression) using standard cloning techniques and expressed in bacteria. Gus was also cloned into Gateway Destination vectors (for native and N-terminal fusion expression) and expressed. When protein expression is compared, there was no difference in the amount of protein produced. This demonstrates that for this particular case, the attB sites do not interfere with transcription or translation.
Effects on solubility: A researcher at the NCI has shown that Maltose Binding Protein fusions constructed with Gateway Cloning were soluble. The fusion proteins expressed had the attB amino acid sequence between the Maltose Binding Protein and the cloned protein. It is possible that some proteins containing the attB sequence could remain insoluble when expressed in E.coli.
Effects on folding: Two Hybrids screens show the same interacters identified with and without the attB sequence. Presumably correct protein folding would be required for protein-protein interactions to take place. It is possible that some proteins containing the attB sequence may not fold correctly.
Since the attB sequences are on the 5' end of oligos, they will not anneal to the target template in the first round of PCR. Sometimes the PCR product is more specific with the attB primers, probably due to the longer annealing sequence (all of attB plus gene specific sequence) after the first round of amplification. Generally there is no need to change PCR reaction conditions when primers have the additional attB sequence
No, this is not really feasible due to the fact that the attL sequence is approximately 100 bp, which is too long for efficient oligo synthesis. Our own maximum sequence length for ordering custom primers is 100 nucleotides. In contrast, the attB sequences are only 25 bp long, which is a very reasonable length for adding onto the 5' end of gene-specific PCR primers.
Vector information can be found in the product manuals or directly on our web site by entering the catalog number of the product in the search box. The vector map, cloning site diagram, and sequence information will be linked to the product page.
The Gateway nomenclature is consistent with lambda nomenclature, but we use numbers to differentiate between modified versions of the att sites (attB1, attB2, attP1, attP2, and so on). We have introduced mutations in the att sites to provide specificity and directionality to the recombination reaction. For example, attB1 will only recombine with attP1 and not with attP2.
The first step is to create an Entry clone for your gene of interest. We have 3 options to do this: The first is by BP recombination reaction using the PCR Cloning System with Gateway Technology. This is recommended for cloning large (>5 kb) PCR products. We also have Gateway compatible TOPO Cloning vectors such as pCR8/GW/TOPO and pENTR/D-TOPO. The final option is to use restriction enzymes to clone into a pENTR Dual Selection vector.
The gene of interest must be flanked by the appropriate att sites, either attL (100 bp) in an Entry clone or attB (25 bp) in a PCR product. For Entry clones, everything between the attL sites will be shuttled into the Gateway destination vector containing attR sites, and a PCR product flanked by attB sites must be shuttled into an attP-containing donor vector such as pDONR221.
The location of translation initiation sites, stop codons, or fusion tags for expression must be considered in your initial cloning design. For example, if your destination vector contains an N-terminal tag but does not have a C-terminal tag, the vector should already contain the appropriate translation start site but the stop codon should be included in your insert.
Yes, increasing the incubation time from 1 hour to 4 hours will generally increase colony numbers 2-3 fold. An overnight incubation at room temperature will typically increase colony yield by 5-10 fold.
BP Clonase II and LR Clonase II can be freeze/thawed at least 10 times without significant loss of activity. However, you may still want to aliquot the enzymes to keep freeze/thaw variability to a minimum.
These enzymes are more stable than the original BP and LR Clonase and can be stored at -20 degrees C for 6 months.
Mini-prep (alkaline lysis) DNA preparations work well in Gateway cloning reactions. It is important that the procedure remove contaminating RNA for accurate quantification. Plasmid DNA purified with our S.N.A.P. nucleic acid purification kits, ChargeSwitch kits, or PureLink kits are recommended.
A simple way to express a protein with a leader sequence is to have the leader sequence encoded in the destination vector. The other option is to have the leader sequence subcloned into the entry vector using restriction enzymes, or incorporate the leader sequence into the forward PCR primer when cloning a PCR product into the entry vector. Please see Esposito et al. (2005), Prot. Exp. & Purif. 40, 424-428 for an example of how a partial leader sequence for secretion was incorporated into an entry vector.
This depends on whether you are expressing a fusion or a native protein in the Gateway destination vector. For an N-terminal fusion protein the ATG will be given by the destination vector and it will be upstream of the attB1 site. For a C-terminal fusion protein or a native protein, the ATG should be provided by your gene of interest, and it will be downstream of the attB1 site.
The Gateway attB sites are derived from the bacteriophage lambda site-specific recombination, but are modified to remove stop codons and reduce secondary structure. The core regions have also been modified for specificity (i.e., attB1 will recombine with attP1 but not with attP2).
Expression experiments have shown that the extra amino acids contributed by the attB site to a fusion protein will most likely have no effect on protein expression levels or stability. In addition, they do not appear to have any effect on two-hybrid interactions in yeast. However, as is true with the addition of any extra sequences that result from tags, the possible effects will be protein-dependent.
No, attB primers are highly specific under standard PCR conditions. We have amplified from RNA (RT-PCR), cDNA libraries, genomic DNA, and plasmid templates without any specificity problems.
The smallest size we have recombined is a 70 bp piece of DNA located between the att sites. Very small pieces are difficult to clone since they negatively influence the topology of the recombination reaction.
There is no theoretical size limitation. PCR products between 100 bp and 11 Kb have been readily cloned into a pDONR Gateway vector. Other DNA pieces as large as 150 kb with att sites will successfully recombine with a Gateway-compatible vector. Overnight incubation is recommended for large inserts.
Standard desalted purity is generally sufficient for creating attB primers. We examined HPLC-purified oligos for Gateway cloning (about 50 bp long) and found only about a 2-fold increase in colony number over standard desalted primers. If too few colonies are obtained, you may try to increase the amount of PCR product used and/or incubate the BP reaction overnight.
Please follow the below steps to find the complete sequence for the Gateway pDONR221 Vector:
- Search "pDONR211" on thermofisher.com
- Filter the results page by "Vector Data" in the left nav
- Once the results are filtered, you can find the restriction, sequence, map, and under and polylinker for the vector under the pDONR211 header.
Find additional tips, troubleshooting help, and resources within our Cloning Support Center.