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DNA marker plasmid and preparation and application thereof

A technology of DNA molecules and plasmids, which is applied in the fields of molecular biology and genetic engineering, can solve problems such as the stability of recombinant plasmids, and achieve the effects of reducing difficulty, improving stability, and improving extraction yield

Active Publication Date: 2013-09-04
生工生物工程(上海)股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the recombinant plasmid constructed in this way has a serious problem, that is, there is a problem with the stability of the recombinant plasmid

Method used

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  • DNA marker plasmid and preparation and application thereof
  • DNA marker plasmid and preparation and application thereof
  • DNA marker plasmid and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Embodiment 1: Construction of DNA marker plasmid

[0075] one. Construct self-circularization parent plasmid M1 (such as SEQ ID NO.1, image 3 )

[0076] The full-length sequence of M1, SEQ ID NO.1, was artificially synthesized, then digested with AflII, and self-circularized to obtain the mother plasmid M1.

[0077] The maternal plasmid was transformed into Escherichia coli DH5a, and it grew well in Amp+ resistant medium ( Figure 4 ), proving that the sequence change in the resistance gene did not affect its function. Meet the experimental requirements.

[0078] two. Construct the full-length recombinant DNA marker plasmid (such as SEQ ID NO.3, Figure 5 )

[0079] The full-length sequence of M2, SEQ ID NO.2, was artificially synthesized, then digested with NdeI / AflII, and cloned between the NdeI / AflII restriction sites of the mother plasmid M1. The full-length recombinant DNA marker plasmid SEQ ID NO.3 was obtained. The results were verified by sequencing, a...

Embodiment 2

[0080] Example 2 Restriction verification of recombinant DNA marker plasmid and preparation of Marker products

[0081] Enzyme digestion verification of recombinant DNA marker plasmid:

[0082] The DNA marker plasmid constructed in Example 1 was respectively digested with SacI, SalI, XhoI, XbaI, EcoRI, BglII, HindIII, KpnI and BamHI to obtain restriction enzyme digestion phenomena A-I.

[0083] Marker product preparation:

[0084] one. Enzyme digestion phenomenon A and B are mixed to obtain a Marker of 100bp to 1000bp (such as Figure 6 BSM0241 shown).

[0085] two. Enzyme digestion phenomenon C and D are mixed to obtain a Marker of 100bp to 2000bp (such as Figure 6 DL2000 shown).

[0086] three. Enzyme digestion phenomenon A, B, D, H, and I are mixed to obtain a Marker of 100bp to 8000bp (such as Figure 6 shown in BSM0258).

[0087] Using different combinations of restriction enzymes, any Marker that meets the requirements can be obtained. The smallest band is 100b...

Embodiment 3

[0088] Example 3 Recombinant plasmid stability verification

[0089] Repeat the transformation of the constructed recombinant DNA marker plasmid several times. The copy number of the plasmid is high, the resistance screening is obvious, and the digestion phenomenon is normal, which proves that the plasmid is very stable.

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Abstract

The invention discloses a DNA marker plasmid and preparation and application thereof. The DNA marker plasmid is a double-chain closed circular DNA molecule, and the nucleotide sequence of the DNA marker plasmid is SEQ ID No:3. Because the high-copy replicon pUC ori is selected, the plasmid extracting rate is improved, and because of high changeability of complete sequence by artificially synthesized, the recombinant DNA marker plasmid does not contain repeat sequences, thereby the plasmid stability is improved and the recombinant DNA marker plasmid can cover the most digestion phenomenon possibilities by the minimum length.

Description

technical field [0001] The invention belongs to the fields of molecular biology and genetic engineering, and specifically relates to a brand-new universal DNA marker plasmid and its preparation and application. Background technique [0002] DNA marker is the most commonly used reagent for indicating the size of DNA fragments, which is essential in genetic engineering experiments. The DNA fragment and the standard DNA marker are electrophoresed in the same agarose gel, and the size of the target DNA fragment can be estimated by comparing the DNA mobility. [0003] Currently, there are two commonly used methods for preparing DNA markers, PCR amplification and plasmid digestion. [0004] PCR amplification is the most commonly used. Although the work is simple, the cost of repetition is high, the amplification efficiency of long fragments is low, and it is necessary to make a reasonable ratio according to the brightness of the product after recovery to achieve the best effect....

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/66C12Q1/68C12N15/11C12N15/10
Inventor 孔凡静王素莲王乾
Owner 生工生物工程(上海)股份有限公司
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