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EXPERIMENT 1
Identification of mouse NEIL cDNA inserts in pGEM-T PCR cloning vectors by
Restriction Endonuclease Mapping.
AIM:
1. To use Restriction Endonucleases and carry out Restriction digestion of pGEM T and
pGEM T Easy vectors containing NEIL cDNA (NEIL1 , NEIL2 and NEIL3) inserts and to
identify the vector and .
2. To record the results for further analysis using restriction endonuclease mapping and
molecular databases.
INTRODUCTION
Procedure :
Step 1:
The first step was to prepare 3 digests. The first Digest is an EcoR1 digest of 3 DNA samples
labelled A1,B1 and C1.This is to help locate which vector the NEIL cDNA has been introduced
into.
The second digest is a double digest containing NdeI and Hind III restriction enzymes.
Labelled A2,B2 and C2.These enzymes will cleave the cDNA insert and reveal which sample
contains NEIL1,NEIL2 and NEIL3.
The third digest contains an extra restriction enzyme Xmn I.Each NEIL cDNA insert contains a
unique Xmn I site.Hence each NEIL cDNA fragment cut is obtained of the same size.
DIGEST 1
3 x 0.5 ml microcentrifuge tubes were obtained and were labelled as A1, B1 and C1.DNA
samples A, B and C were transferred to tubes A1, B1 and C1 respectively.The reagents for the
preparation of the restriction were prepared (including DNA samples) according to the
following formula.
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Using a P20 pipette, reagents for restriction digest along with corresponding DNA samples (A
, B and C) were transferred to respective microcentifuge tubes. The mixture in the tubes
was homogenized by vortexing briefly for about 1-2 seconds allowing the centrifuge to reach
a speed of ~5000rpm.The tubes were then incubated for 1h at 37*C in a water bath.
DIGEST 2
As similar to procedure in DIGEST 1, 3 x 0.5 ml microcentrifuge tubes were obtained and
labelled as A2, B2 and C2.Corresponding DNA samples for the 3 tubes with restriction digest
mixture were transferred. They were then centrifuged till the centrifuge reaches ~5000rpm
and then incubated for 1 hour in a water bath at 37*C.
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DIGEST 3
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Step 2:
0.24g of agarose was weighed and transferred into a 250ml conical flask and 30ml of 0.5X
TBE buffer was added to it. The flask was plugged with a piece of cotton wool and heated in
short bursts (30 seconds) by microwaving on medium setting till the mixture boils and
agarose has fully dissolved.
The agarose solution was allowed to cool to 55*C and 30 micro Litres of Gel Red was added.
The Flask was shaken and swirled to ensure uniform mixing.The gel was then poured into the
gel tray and a comb was inserted to create wells and then left aside to set.
Step3
The tubes were removed after 1h from the water bath and pulse spun to remove any sort of
condensation that might have formed on the lid of the tube.
The comb was removed and 3.0 micro Litres of loading buffer was added to each sample and
mixed. To WELL 1, 10micro Litres of DNA size marker was added. The prepared samples were
then added in order of A1,B1 & C1; A2,B2 & C2 and A3,B3 and C3.
Step4:
With the wells facing the negative terminal of the gel electrophoresis apparatus and lid in
place, a voltage of 100v was delivered. Making sure current is flowing, it was allowed to run
for 45-50 min.
Once the time interval has lapsed, the gel was placed on a UV-transilluminator to visualise
DNA bands. A picture was then taken for analysis.
RESULT
A1 B1 C1 A2 B2 C2 A3 B3 C3
OBTAINED RESULT
CONTROL
In A1,B1 and C1, 2 bands are observed in C1 while only 1 band is observed in A1 and B1.Hence EcoR1
site is present in C1.Thus it can be concluded that A1 and B1 are pGEM-T vectors while C1 contains
pGEM-T vector.
In digest 2, the fragment separated at A2 is the longest one separated.Hence the fragment separated
at A2 can be NEIL3 which is the longest in base pairs.Well C2 is NEIL1 because it is the second
longest.The third at B2 separated the least towards the negative terminal and thus it is NEIL2.In
digest 3 , specific sized fragments of NEIL1,NEIL2 and NEIL3 were obtained.Their sizes were
calculated as follows
Calculation
Band 1
NEIL I size= 707
EXPERIMENT 2
Analysis of DNA Sequences using Restriction Mapping and Molecular
Databases.
AIM:
4. To determine the identity of PCR cloning vector for each DNA sample.
NEBcutter(http://tools.neb.com/NEBcutter2/index.php
The NCBI or National Centre for Biotechnology information is the largest and most
comprehensive compilation of publicly accessible DNA sequences, biomedical and
genomic information and molecular biology information.
Procedure
To carry out the restriction mapping on the pGEM-NEIL cDNA samples, mRNA nucleotide sequences
for NEIL1,NEIL2 and NEIL3 have to be obtained.These mRNA sequences can be obtained from NCBI’s
molecular gene database.
The following url was typed into the browser and navigated to.
http://www.ncbi.nlm.nih.gov/
For search query, NEIL1 was entered and was searched for under ‘Gene’.The following results were
displayed.
In the search results, the third entry is for NEIL1 [Mus musculus].The result was opened by clicking on
it.
Restriction mapping was carried out by navigating to web mapper hosted by Harvard Medical school
at:
<http://pga.mgh.harvard.edu/web_apps/web_map/start >
The sequence for NEIL1 was copied into the box and was submitted.
Xmn I site for NEIL1:
707 472
Similarly the CDS for NEIL2 and NEIL3 was found out
569 421
BLAST:
Its is Basic Local Alignment Search Tool.To obtain more information on the homology
between NEIL proteins, BLAST has to be run.
http://blast.ncbi.nlm.nih.gov/Blast.cgi
To check if there is any homology in nucleotide sequence of bacterial with NEIL proteins,a
search was conducted for ‘nei and e cole’.The following is the result.
Clicking NC_000913.2 and choosing the FASTA format, the following DNA sequence of nei gene was
obtained....
The sequence was then pasted into BLASTN and was searched.
BLAST SEARCH
The “super families” box in the graphical summary reveals us more information regarding conserved
domain.
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