![SMOBIO/[CV1100] GetClone™ PCR Cloning Vector II, 20 Rxn/s)"}]]>
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Description
The GetClone™ PCR Cloning Vector II is a positive selection system for high efficiency cloning of blunt end DNA or amplicons. This cloning vector contains a lethal gene which can be disrupted by ligation of a blunt end DNA insert into the cloning site. Only colonies with inserted vectors are able to propagate, eliminating the additional needs of IPTG and X-Gal for blue/white screening. This cloning vector includes ampicillin and kanamycin resistance genes that can meet the needs of most users.
Features
Cloning efficiency greater than 90%
IPTG and X-Gal are not required
Accepts a wide range of insert/vector ratios 0.5:1 to 12:1
Accepts insert size from 6 bp to 11 kb
The phosphorylation of PCR fragments is not required
Accepts blunt end amplicon or DNA fragment (not for sticky ends)
Ampicillin and kanamycin selection markers
Storage
-20°C for 24 months
The plasmid map of pGet II Vector
The plasmid cloning sites of pGet II Vector
Contents
Component | Volume |
pGet II Vector (25 ng/μl) | 23 μl |
pGet-For Primer (10 μM) | 100 μl |
pGet-Rev Primer (10 μM) | 100 μl |
Primers Sequence
pGet-For Primer:
5"-TCGAAGTTAAAGATGATTACGG-3"
pGet-Rev Primer:
5"-TCTCTCGATAGCATTTCCTGC-3"
Storage
-20°C for 24 months
Manual
Manual_CV1100_GetClone™ PCR Cloning Vector II
Sequence in Fasta format
Sequence_CV1100_GetClone™ PCR Cloning Vector II
SDS
SDS_CV1100
Why use the GetClone™ Vector for PCR cloning instead of a T-vector?
For conducting PCR-cloning, a high fidelity polymerase is required to keep accuracy of PCR amplification. In general, high fidelity enzymes vetting correctness such as SMO-HiFi™, Pfu, KOD, and Phusion are associated with proofreading activity which results in blunt ends of PCR products. GetClone™ Vector can directly accept blunt-ended PCR fragments for ligation, without the need of extra step of DNA phosphorylation. As contrast, T-vector used for TA-cloning requires DNA fragment with an extra Adenosine nucleotide added at the 3’ end which is usually generated by non-proofreading and thus less fidelity DNA polymerase like Taq polymerase.
Is blunt end ligation more difficult than sticky end ligation?
It depends on the length of sticky end. Generally, sticky end is easier to perform in comparison to blunt end ligation. However, a sticky end with only one extruding base as seen in the TA-cloning is probably more difficult for ligation as compared with a blunt end (also referred to NEB for NciI or EcoNI instructions).
After using GetClone™ Vector for PCR cloning, how can the colonies carrying the cloned fragment be selected?
In most cases of using GetClone™ Vector, colonies grown on a plate with appropriate antibiotics should be the positive clones because a bacterium accepting an empty GetClone™ Vector without an insert would fail to proliferate. To further identify the clones, it is recommended to use Colony-PCR method. In brief, the colony grown on the plate is directly picked up with a toothpick and then mixed into a PCR premix (ex.: SMOBIO TP1200 or TP1260) and PCR amplification is conducted in the thermal cycler with primers (pGET-For and pGET-Rev) which are also provided with GetClone™ Vector. The expected length of PCR product should be the one of the insert plus 152 bp.
How do I perform sequencing when using the GetClone™ Vector?
Please use the GetClone™ Vector primers for sequence service.
In use of GetClone™ Vector for cloning, why are some colonies in absence of inserts?
Several reasons can be stated as follows:
Reason 1: Check your insert. If it is not blunt-end, it cannot be ligated with GetClone™ vector, as this may result in false positive results for majority of the colonies.
Reason 2: When DNA concentration of the insert is too low, the successful ligates are largely reduced in proportion, leading to an overwhelming vector background. The solution is to increase the concentration of the insert DNA.
Reason 3: If the target DNA fragments for cloning are large, such as 10kb or more, transformation efficiency depreciates after ligation. This results in fewer colonies having insertions. Therefore, it is suggested to increase the insert DNA concentration and reduce the concentration of vector DNA to re-execute ligation. It will also enhance chances to get the right clone by using higher efficiency competent cells.
Reason 4: Non-specific PCR products or/and primer dimers which might occurred during PCR amplification are supposed to compete or interfere the ligation of target PCR fragment with the vector, and thus reducing the number of correct clones. Before conducting ligation, it is therefore recommended to isolate the desired DNA fragment from other non-specific DNA by gel extraction.
If I did not add any insert DNA fragment for ligation with GetClone™ Vector, why are colonies being grown on the selective plate?
This is probably due to generation of mutation on the vector DNA during cloning. A mutated lethal gene can lead to false positive colonies, despite that the occurrence is rare.
How to count the 90% cloning efficiency of GetClone™?
The GFP report gene was PCR amplified as the insert to the GetClone™ Vector. After ligation and transformation, positive clones (with insert) presenting green fluorescence on the plate were calculated in number, and ~ 90% of total colonies did shine as observed in B-Box, demonstrating the high cloning efficiency of GetClone™ Vector .
Do you have any experiment data for cloning 12 kb fragment with GetClone™ Vector?
Yes, we do have the data as shown below. Eight colonies were picked up for plasmid isolation and PCR analysis. It was observed that seven out of eight colonies (87.5%) were positive clones which could be verified by successful amplification of 12.1 kb fragments.
Ligation Example 1 (NEB T4 DNA Ligase #M0202)
Insert (Blunt end) X μl (Y ng*)
pGet II (3954 bp) 1 μl (25 ng)
Mix well then add
10X T4 DNA Ligase Buffer 2 μl
T4 DNA Ligase 1 μl
ddH2O to 20 μl
Final volume 20 μl
Mix well then incubate at 16°C or room temperature (20~25°C) for 1 hours.
Ligation Example 2 (TOYOBO Ligation High ver2 #LGK-201)
Insert (Blunt end) X μl (Y ng*)
pGet II (3954 bp) 1 μl (25 ng)
ddH2O up to 7 μl
Ligation high ver2 3.5 μl
Final volume 10.5 μl
Mix well then incubate at 16°C or room temperature (20~25°C) for 5~30 mins.
*For 3/1 of Insert/Vector molar ratio:
Transformation
The GetClone™ is compatible with most available competent E. coli cells. Apply 1 ~10 µl of ligation mixture to 10 times volume competent E. coli cells. Perform transformation procedures according to the instruction of the competent cells. Spread the transformed E. coli cells on an LB-ampicillin (50~100 µg/ml) or LB-Kanamycin (50 µg/ml) plate for colony selection.
Recommended colony PCR condition
(SMOBIO’s TP1200 ExcelTaq™ 5X PCR Master Dye Mix is suggested)
Template | Single colony |
pGet-For Primer | 0.5 µl |
pGet-Rev Primer | 0.5 µl |
5X PCR Master Mix | 5 µl |
H2O | to 25 µl |
Total volume | 25 µl |
Recommended PCR Program
Steps | Temp. | Time | Cycles |
Template denature | 94°C | 2 min | 1 |
Denature | 94°C | 30 sec | 25-40 |
Annealing | 50°C | 30 sec | |
Extension | 72°C | 30 sec/kb | |
Final extension | 72°C | 1 min | 1 |
High Fidelity PCR amplification
Amplification of target gene with HiFi™ DNA polymerase to minimize error rate.
[TF1000] SMO-HiFi™ DNA Polymerase, (1 U/μl, 100 U)
[TF3000] G-HiFi™ DNA Polymerase, (1 U/μl, 100 U)
Gel electrophoresis
Staining amplicons with safe fluorescent dyes, following by observation under blue-light illuminator to minimize damage of DNA amplicons and maximize successful cloning efficiency.
Safe fluorescent dyes
[NS1000] FluoroVue™ Nucleic Acid Gel Stain (10,000X), 500 μl
[DS1000] FluoroStain™ DNA Fluorescent Staining Dye (Green, 10,000X), 500 μl
[DL5000] FluoroDye™ DNA Fluorescent Loading Dye (Green, 6X), 1 ml
Blue-light illuminator
[VE0100] B-BOX™ Blue Light LED Epi-illuminator
Transformation
Prepare competent cells with high efficiency and transform with time-saving protocol.
[CK1000] Champion™ E. coli Transformation Kit
Colony PCR
Analyze colonies with PCR master mix to save preparation time.
[TP1100] ExcelTaq™ 5X PCR Master Mix, 200 Rxn
[TP1200] ExcelTaq™ 5X PCR Master Dye Mix, 200 Rxn
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准备RNA用的试剂：70%乙醇（用DEPC处理后的水稀释新开封的无水乙醇），DEPC处理后的水，新开封的三氯甲烷等，
容器：玻璃试剂瓶需要180度，8个小时以上烘烤
Eppendorf管和“枪头”：RNase-free的管子和“枪头”（可以直接买到）；或者普通的ep管和“枪头”，但是需要DEPC水浸泡后，高温灭菌（121度，20-40分钟）。
如果需要使用研钵和药品匙，研钵和药品匙也是需要180度，8小时以上的烘烤。
还需要没开封的手套，提取RNA时，尽量勤换手套，少说话，尽量不要对着样品吹气。
总RNA提取试剂盒（TRIzol法）
RIpure试剂是直接从细胞或组织中提取总RNA的试剂。它在破碎和溶解细胞时能保持RNA的完整性。加入氯仿后离心，样品分成水样层和有机层。RNA存在于水样层中。收集上面的的水样层后，RNA可以通过异丙醇沉淀来还原。在除去水样层后，样品中的DNA和蛋白也能相继以沉淀的方式还原。乙醇沉淀能析出中间层的DNA，在有机层中加入异丙醇能沉淀出蛋白。共纯化DNA对于样品间标准化RNA的产量十分有用。
无论是人、动物、植物还是细菌组织，该方法对少量的组织(50-100mg)和细胞(5×106)以及大量的组织(≥1g)和细胞(>107)均有较好的分离效果。TRIPURE试剂操作上的简单性允许同时处理多个的样品。所有的操作可以在一小时内完成。TRIPURE抽提的总RNA能够避免DNA和蛋白的污染。故而能够作RNA印迹分析、斑点杂交、poly(A)+选择、体外翻译、RNA酶保护分析和分子克隆。如果是用于PCR，当两条引物位于单一外显子内时，建议用扩增级的DNase I来处理抽提的总RNA。
TRIpure试剂能促进不同种属不同分子量大小的多种RNA的析出。例如，从大鼠肝脏抽提的RNA琼脂糖凝胶电泳并用溴化乙啶染色，可见许多介于7 kb和15 kb之间不连续的高分子量条带(mRNA和hnRNA成分)，两条优势核糖体~5 kb (28S)和~2 kb(18S)，低分子量RNA介于0.1和0.3 kb之间 (tRNA, 5S)。当抽提的RNA用TE稀释时其A260/A280比值≥1.8。
Trizol法和试剂盒提取组织中的RNA，哪个方法更可靠和精确呢？谢谢！
RNA提取对于科研人员来说并不陌生，但是要得到好的结果却不是一件很容易的事情。事实上，现在市面上的丰富的RNA提取试剂基本上可以满足科研人员的需要，为什么往往提取的RNA却容易失败呢？
RNA提取失败的主要现象有三个：提取的RNA降解、提取的RNA量偏低以及提取的RNA纯度低。究竟怎样才能确保RNA提取的成功率呢？
首先，要确定材料的可用性。尽管现有的RNA提取试剂都用抑制RNase的成分，但提取的材料仍需谨慎处理。提取的材料的新鲜度是获取完整RNA的关键，但是由于种种原因无法立即从新鲜材料中提取RNA时，使用Takara公司的样品保护剂SampleProtectorforRNA/DNA可以免去使用液氮或超低温冰箱的不便，同时也可以有效解决组织、细胞样品的短时间保存及运输问题。此外，如果将不同时期收集的样品都预先存放于本试剂中，可以做到立即终止并固定RNA表达的时序变化，减少实验组间的误差。
其次，选择合适的提取试剂是最重要的一步。好的提取试剂在确保成功的同时，操作方便且经济实用。对于动物组织、简单的植物材料、各种微生物、培养细胞的totalRNA提取，Takara公司的RNAisoPlus具有诸多的成功案例。随着2013年7月柱型提取试剂TaKaRaMiniBESTUniversalTotalRNAExtractionKit的成功上市，进一步丰富了TakaraRNA提取的产品线。该产品采用了独特的细胞裂解系统，无需苯酚氯仿抽提等步骤，简单快捷。组织或细胞裂解后，提取操作仅需20分钟便可完成。
对于富含多糖多酚的植物类组织提取是个难点，Takara精心研发的柱型提取试剂TaKaRaMiniBESTPlantRNAExtractionKit可以轻松解决这个问题。TaKaRaMiniBESTPlantRNAExtractionKit可以高效地从各种简单的植物组织材料（叶片、茎、幼苗等）、富含多糖多酚的植物组织材料（果实、种子等）、真菌提取高纯度的TotalRNA，适用范围广泛。按照标准流程，本试剂盒可以有效地提取分子量大于200nt的RNA，也可以按照可选步骤提取得到包含SmallRNA(<200nt)的TotalRNA。试剂盒中包含了RNA提取所需的全部试剂，无需额外购买其它试剂。
动物体内RNA转染试剂，核酸和转染试剂混合后注射，轻松进行RNA干扰，基因敲除、沉默实验，3天可得出结果。下面以50μg的核酸与25μl转染试剂，总注射体积200μl，20g小鼠尾静脉注射为例说明。局部注射不用稀释，直接根据需要把核酸和转染试剂混合即可使用。
1.核酸的稀释。将50μg核酸用适量无内毒素纯水稀释成1μg/μl，加入10%葡萄糖溶液(w/v)50μl，使葡萄糖终浓度为5%，终体积为100μl，充分混匀。
2.转染试剂的稀释。取25μl的Entranster-invivo试剂用50μl的10%葡萄糖溶液稀释，并用纯水25μl补足，得到葡萄糖终浓度为5%，终体积为100μl液体，充分混匀。
3.转染复合物形成。立即将稀释后的转染试剂加入到稀释后的核酸溶液中，立即充分振荡混匀。
4.室温静置15分钟。配制好的转染复合物请即配即用，不宜长期存放。
5.动物注射。
说明：1).尾静脉注射时请掌握注射技巧，一般选用远端1/3处静脉注射，如感觉到阻力和轻微隆起，请停止注射，重新寻找静脉进行操作，不要强力推注，否则容易将药液注射在尾部，导致尾部溃烂。注射完成后移去针头，按压针孔10秒以上，防止药液流出。2).2.5mg/kg的给药剂量是起始给药剂量，如果动物可以耐受，可以按比例增加剂量，这样效果更好。3).局部注射，尽可能多注射药液，有利于提高转染效果。
6.基因表达检测。一般来说，根据注射方法和靶器官的差异，12-48小时后基因表达效果较好。
7.长期给药。一次给药检测的最佳时间是注射后12-48小时。如果需要维持长期效果，可以采用多次注射的方法，注射频度一般为每间隔2-3天一次，也可以根据实验情况适当延长至每7天一次。
氯仿萃取RNA
异丙醇沉淀RNA
酒精清洗
