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Straightforward procedure for laboratory production of DNA ladder.

Lan VT, Loan PT, Duong PA, Thanh le T, Ha NT, Thuan TB - J Nucleic Acids (2012)

Bottom Line: DNA fragments of 100 bp with unique restriction site at both ends were self-ligated to create a tandem repeat.Once being cloned, the tandem repeat was rapidly amplified by PCR and partially digested by restriction enzymes to produce a ladder containing multimers of the repeated DNA fragments.Our procedure for production of DNA ladder could be simple, time saving, and inexpensive in comparison with current ones widely used in most laboratories.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Biology, Hanoi University of Science, Vietnam National University, 334 Nguyen Trai Street, Thanh Xuan, Hanoi, Vietnam.

ABSTRACT
DNA ladder is commonly used to determine the size of DNA fragments by electrophoresis in routine molecular biology laboratories. In this study, we report a new procedure to prepare a DNA ladder that consists of 10 fragments from 100 to 1000 bp. This protocol is a combination of routinely employed methods: cloning, PCR, and partial digestion with restriction enzymes. DNA fragments of 100 bp with unique restriction site at both ends were self-ligated to create a tandem repeat. Once being cloned, the tandem repeat was rapidly amplified by PCR and partially digested by restriction enzymes to produce a ladder containing multimers of the repeated DNA fragments. Our procedure for production of DNA ladder could be simple, time saving, and inexpensive in comparison with current ones widely used in most laboratories.

No MeSH data available.


Related in: MedlinePlus

Production of 100 bp DNA ladder. (a) The pGEM-800 plasmid was partially digested by SmaI enzyme producing 8 fragments in length of 100–800 bp. (b, c) Electrophoresis of the prepared 100 bp DNA ladder. A 5 μL (lane 2) or 3 μL (lane 3) of DNA ladder prepared in the present study and 100 bp DNA marker from Takara (M) were submitted to 2% agarose (b) or 12% acrylamide (c) gel electrophoresis, respectively.
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fig2: Production of 100 bp DNA ladder. (a) The pGEM-800 plasmid was partially digested by SmaI enzyme producing 8 fragments in length of 100–800 bp. (b, c) Electrophoresis of the prepared 100 bp DNA ladder. A 5 μL (lane 2) or 3 μL (lane 3) of DNA ladder prepared in the present study and 100 bp DNA marker from Takara (M) were submitted to 2% agarose (b) or 12% acrylamide (c) gel electrophoresis, respectively.

Mentions: Our study successfully produced 100 bp DNA ladder with 10 fragments ranging from 100 to 1000 bp. Our procedure contained three steps. In detail, the first step was to make a 100 bp DNA fragment from a known sequence by using two specific primers that contain restriction site at the 5′ ends. One fragment out of sequenced ones in our experiments that contains SmaI recognition site at the 5′ end was cloned; thus, we did make the specific primers Sma-100F and Sma-100R. The selection of sequences and specific primers for preparation of 100 bp DNA fragment is easy and flexible in most laboratories working on DNA. The second step was cloning PCR products which were reamplified from self-ligated DNA of SmaI-digested 100 bp fragments. Using universal primers of pGEM-T vector, the largest insert of 800 bp was quickly selected, and recombinant plasmid containing this fragment was named pGEM-800. It should be noted that some inserts larger than 800 bp could be selected when the self-ligated reaction was performed by commercial DNA ligation kits with a special efficiency in DNA ligation. Thus, a 100 bp DNA ladder with a range outside of 1000 bp could be generated. Since the same unique SmaI restriction site lies at each junction of the 100 bp repeat units, the pGEM-800 could be partially digested by this enzyme to produce a 100 bp DNA ladder (Figure 2(a)). In this case, our DNA ladder contained 8 fragments ranging from 100 to 800 bp. We observed that a large amount of pGEM-800 plasmid is required for clear visibility of small fragments as 100 bp–200 bp. In addition, partial digestion of plasmids was dependent on plasmid conformation (supercoilled, circular and linear forms); thus, it was not easy to reproduce.


Straightforward procedure for laboratory production of DNA ladder.

Lan VT, Loan PT, Duong PA, Thanh le T, Ha NT, Thuan TB - J Nucleic Acids (2012)

Production of 100 bp DNA ladder. (a) The pGEM-800 plasmid was partially digested by SmaI enzyme producing 8 fragments in length of 100–800 bp. (b, c) Electrophoresis of the prepared 100 bp DNA ladder. A 5 μL (lane 2) or 3 μL (lane 3) of DNA ladder prepared in the present study and 100 bp DNA marker from Takara (M) were submitted to 2% agarose (b) or 12% acrylamide (c) gel electrophoresis, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3306978&req=5

fig2: Production of 100 bp DNA ladder. (a) The pGEM-800 plasmid was partially digested by SmaI enzyme producing 8 fragments in length of 100–800 bp. (b, c) Electrophoresis of the prepared 100 bp DNA ladder. A 5 μL (lane 2) or 3 μL (lane 3) of DNA ladder prepared in the present study and 100 bp DNA marker from Takara (M) were submitted to 2% agarose (b) or 12% acrylamide (c) gel electrophoresis, respectively.
Mentions: Our study successfully produced 100 bp DNA ladder with 10 fragments ranging from 100 to 1000 bp. Our procedure contained three steps. In detail, the first step was to make a 100 bp DNA fragment from a known sequence by using two specific primers that contain restriction site at the 5′ ends. One fragment out of sequenced ones in our experiments that contains SmaI recognition site at the 5′ end was cloned; thus, we did make the specific primers Sma-100F and Sma-100R. The selection of sequences and specific primers for preparation of 100 bp DNA fragment is easy and flexible in most laboratories working on DNA. The second step was cloning PCR products which were reamplified from self-ligated DNA of SmaI-digested 100 bp fragments. Using universal primers of pGEM-T vector, the largest insert of 800 bp was quickly selected, and recombinant plasmid containing this fragment was named pGEM-800. It should be noted that some inserts larger than 800 bp could be selected when the self-ligated reaction was performed by commercial DNA ligation kits with a special efficiency in DNA ligation. Thus, a 100 bp DNA ladder with a range outside of 1000 bp could be generated. Since the same unique SmaI restriction site lies at each junction of the 100 bp repeat units, the pGEM-800 could be partially digested by this enzyme to produce a 100 bp DNA ladder (Figure 2(a)). In this case, our DNA ladder contained 8 fragments ranging from 100 to 800 bp. We observed that a large amount of pGEM-800 plasmid is required for clear visibility of small fragments as 100 bp–200 bp. In addition, partial digestion of plasmids was dependent on plasmid conformation (supercoilled, circular and linear forms); thus, it was not easy to reproduce.

Bottom Line: DNA fragments of 100 bp with unique restriction site at both ends were self-ligated to create a tandem repeat.Once being cloned, the tandem repeat was rapidly amplified by PCR and partially digested by restriction enzymes to produce a ladder containing multimers of the repeated DNA fragments.Our procedure for production of DNA ladder could be simple, time saving, and inexpensive in comparison with current ones widely used in most laboratories.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Biology, Hanoi University of Science, Vietnam National University, 334 Nguyen Trai Street, Thanh Xuan, Hanoi, Vietnam.

ABSTRACT
DNA ladder is commonly used to determine the size of DNA fragments by electrophoresis in routine molecular biology laboratories. In this study, we report a new procedure to prepare a DNA ladder that consists of 10 fragments from 100 to 1000 bp. This protocol is a combination of routinely employed methods: cloning, PCR, and partial digestion with restriction enzymes. DNA fragments of 100 bp with unique restriction site at both ends were self-ligated to create a tandem repeat. Once being cloned, the tandem repeat was rapidly amplified by PCR and partially digested by restriction enzymes to produce a ladder containing multimers of the repeated DNA fragments. Our procedure for production of DNA ladder could be simple, time saving, and inexpensive in comparison with current ones widely used in most laboratories.

No MeSH data available.


Related in: MedlinePlus