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Božena Pálinkášová
bozena.palinkasova@student.mmu.ac.uk

Dostupný celý text práce:
DOC
r. 2007

Školiteľ:
Mr H N Hughes (BA, BSc, DipRCPath)

Kľúčové slová:
in vivo cloning, pUC18

Vedný odbor:
PRÍRODNÉ VEDY » Biologické vedy » Všeobecná biológia

Škola:
Univerzita mimo Slovenska

Dublin Core:
Dublin Core verzia

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Verzia vo formáte MARC21XML

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Verzia pre Iniciatívu otvorených archívov

Upozornenie: obsah diplomovej práce je chránený autorským zákonom č. 618/2003 Z.z.

Investigations into the Lambda / HindIII Restriction Fragment Insertion Frequencies Using pUC18 as a Cloning Vector

Božena Pálinkášová (Školiteľ: Mr H N Hughes (BA, BSc, DipRCPath)) | pridané: 7. júla 2007

Abstrakt bakalárskej práce:

Plasmids are essential tools in the molecular biology due to having a number of advantageous properties. They are easy to manipulate and their covalent closed circular form makes them easy to differentiate and purify from the linear genomic DNA. They are extremely useful in molecular cloning, as they replicate independently and produce up to 500 copies per cell.

In this project, frequencies of the lambda DNA / HindIII restriction fragments cloned into pUC18 were investigated. Eight lambda DNA restriction fragments produced by HindIII vary in size, ranging from 125bp – 23,130bp; therefore comparison of these fragments of different sizes can be made. The plasmid was cut with HindIII creating a complementary ‘sticky ends’, ligated at random with a mix containing eight lambda fragments and transformed to Escherichia coli. Cells were allowed to replicate and produce clones of the recombinant plasmid. After incubation for 24 hours, the recombinant plasmid DNA was extracted and analysed.

Results showed that half of the lambda fragments failed to be inserted and cloned into the pUC18, which was probably due to being considerably larger in comparison with the size of the vector. The remaining fragments that were successfully cloned were statistically analysed and the frequencies of the occurance were highly significantly different.

During the project, the actual number of lambda restriction fragments produced by HindIII was investigated due to contradictory literature statements. It was revealed that wild-type lambda DNA has six restriction sites for HindIII and a standard lambda laboratory strain (cIind 1 ts857Sam7), which used for this experiment, has seven HindIII restriction sites due to a single base substitution.

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Bibliografický odkaz

PÁLINKÁŠOVÁ, Božena: Investigations into the Lambda / HindIII Restriction Fragment Insertion Frequencies Using pUC18 as a Cloning Vector [ Bakalárska práca ] Univerzita mimo Slovenska. Školiteľ: Mr H N Hughes (BA, BSc, DipRCPath). Rok obhajoby: 2007

Bachelor Thesis:

nvestigations into the Lambda / HindIII Restriction Fragment Insertion Frequencies Using pUC18 as a Cloning Vector

Božena Pálinkášová (Supervisor: Mr H N Hughes (BA, BSc, DipRCPath)) | added: 7. júla 2007

Abstract of bachelor thesis:

Plasmids are essential tools in the molecular biology due to having a number of advantageous properties. They are easy to manipulate and their covalent closed circular form makes them easy to differentiate and purify from the linear genomic DNA. They are extremely useful in molecular cloning, as they replicate independently and produce up to 500 copies per cell.

Plasmids are essential tools in the molecular biology due to having a number of advantageous properties. They are easy to manipulate and their covalent closed circular form makes them easy to differentiate and purify from the linear genomic DNA. They are extremely useful in molecular cloning, as they replicate independently and produce up to 500 copies per cell.

In this project, frequencies of the lambda DNA / HindIII restriction fragments cloned into pUC18 were investigated. Eight lambda DNA restriction fragments produced by HindIII vary in size, ranging from 125bp – 23,130bp; therefore comparison of these fragments of different sizes can be made. The plasmid was cut with HindIII creating a complementary ‘sticky ends’, ligated at random with a mix containing eight lambda fragments and transformed to Escherichia coli. Cells were allowed to replicate and produce clones of the recombinant plasmid. After incubation for 24 hours, the recombinant plasmid DNA was extracted and analysed.

Results showed that half of the lambda fragments failed to be inserted and cloned into the pUC18, which was probably due to being considerably larger in comparison with the size of the vector. The remaining fragments that were successfully cloned were statistically analysed and the frequencies of the occurance were highly significantly different.

During the project, the actual number of lambda restriction fragments produced by HindIII was investigated due to contradictory literature statements. It was revealed that wild-type lambda DNA has six restriction sites for HindIII and a standard lambda laboratory strain (cIind 1 ts857Sam7), which used for this experiment, has seven HindIII restriction sites due to a single base substitution.