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DNA Ligation and Transformation

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Construction of recombinant DNA molecules through DNA ligation and transformation is a very important basic tool in molecular biology. Transformation is used for cloning or to move DNA molecules around between strains. Bacteria are transformed for numerous different reasons. It includes expression of medically useful recombinant proteins such as insulin for treating a disease or vaccines for prevention of disease.

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									DNA Ligation and Transformation

Introduction :

       Construction of recombinant DNA molecules through DNA ligation and transformation
is a very important basic tool in molecular biology. Transformation is used for cloning or to
move DNA molecules around between strains. Bacteria are transformed for numerous different
reasons. It includes expression of medically useful recombinant proteins such as insulin for
treating a disease or vaccines for prevention of disease.

       In this practical, the objective is to clone the DNA fragment encoding mouse beta-actin
into the pGEM-T vector, followed by the transformation of ligated recombinant DNA molecules
into the E.coli strain DH5α . The uptake plasmid DNA is further isolated from E.coli , selected
against , verified by enzyme digestion , and finally visualized and analysed by gel
electrophoresis.

Materials :

2X Rapid Ligation Buffer for T4 DNA ligase , Linearized pGEM-T vector , PCR product ,
Control Insert DNA, T4 DNA ligase , Deionized water, SOC plate , Wizard miniprep Promega
kit , Solution A , Solution B, Alkaline protease solution, Solution C, Solution D , 10X Digestion
buffer , NcoI restriction enzyme, Sal I restriction enzyme , 1.2% agarose gel in TBE , Cyber Safe,
10X loading Dye, 1kb Ladder.




Procedures:

       The PCR product ( mouse beta actin ) was ligated and incubated at room temperature
with pGEM-T vector in standard ligation reaction , with an extra positive and an extra
background ligation reaction as control . The E.coli DH5 α strain was made competent by ice
treatment , and was added to the ligation mixture follow by a brief heat shock and incubation.
The suspension of standard reaction , positive reaction and background reaction were cultured
separately on SOC plate , incubated overnight at 37 C , and the number of blue and white
colonies were tabulated. Each single blue colony and single white colony from Standard
Reaction was transferred and cultured overnight at 37C in SOC plate. The recombinant plasmids
were isolated from E.coli by a small scale preparation protocol using Wizard miniprep kit from
Promega and stored. Each plasmid from blue and white colony were digested by NcoI or/and
SalI restriction enzyme following by incubation at 37C for an hour. The vector pGEM-T and the
insert was separated using Agarose gel electrophoresis and visualized under UV light box.




Results:

                    Table 1: Colony amount of different ligation reaction

                                                      Colony
Ligation Reaction                        White                   Blue
Standard Reaction                        2                       0
Positive Control                         0                       1
Background Control                       0                       0



Table 2: Concentration and absorption ratio of purified recombinant plasmid DNA

Plasmid form                  Concentration (μL/ng)            Absorption Ratio 260/280
White Colonies                101.5                            1.88
Blue Colonies                 98.5                             1.88


*Normal range of recombinant plasmid DNA concentration was yield.
*Lane 1: Uncut plasmid from blue colony
 Lane 2: Nco-I digested plasmid from blue colony
 Lane 3: SalI digested plasmid from blue colony
 Lane 4 : NcoI and SalI digested plasmid from blue colony
 Lane 5 :Uncut plasmid from white colony
 Lane 6: Nco-I digested plasmid from white colony
 Lane 7: SalI digested plasmid from white colony
 Lane 8 : NcoI and SalI digested plasmid from white colony
 Lane 9 : DNA 1kbp ladder


              Figure : Digital image of plasmid restriction digests on 1kbp ladder




Discussion:
Blue white selection

To select the transformed bacterial cell from the plate that have taken up the recombinant
plasmid, the antibiotic (Amphicilin ) and a-complementation of β galactosidase is used. The
antibiotic Amphicilin is a selection for antibiotic resistant colonies which have uptake the
amphicilin resistant gene in vector plasmid. Thus, only cell with uptake of plasmid would be
antibiotic resistant and survived as white colony. The β-galactosidase is a selection of bacteria
colonies which have insertion of the DNA into plasmid which form recombinant plasmid. The
recombinant plasmid with insertion of DNA would have disrupted Lac Z N-terminal , thus
unable to have α-complementation with E.coli cell which expressing Lac Z C-terminal. The
colony will remained white since no active b-galactosidase is produced to cleave the X-gal to
produce blue product.

Hence, white colony is observed for the cell that have the uptake of recombinant plasmid, blue
colony is observed for the cell which have the uptake of non-recombinant plasmid, and no
colony will be observed for cell which have no uptake of plasmid.

From the result of table, the low amount/absence of white colony in standard reaction and
positive control is due to the poor result of transformation. Unequal mixing of the ligation
mixture with the E.coli cell during pipetting causing only small area of contact between the cell
and the plasmid for transformation. This shows that uniform mixing of the reaction is important
to facilitate the uptake of plasmid into E.coli cell.

Restriction digestion analysis and gel electrophoresis

From the result of figure 1 , in general, the bright band of 3kb is observed from the uncut
plasmid and the plasmid with single restriction digestion . NcoI or SalI is only having one cut
side in the plasmid thus producing only one 3kb band of vector plasmid.

The band from lane 1-4( plasmid from blue colony) is slightly lower than the band from 5-8
( plasmid from white colony ). This shows that the migration speed of plasmid from blue colony
is slightly faster than plasmid of white colony , due to the difference in nucleotide base pair size .
The plasmid of white colony which containing the recombinant DNA will have higher base pair
size (>3kb) as compared to the plasmid of blue colony , thus will be having lower migration
speed.

Specifically, lane 1 and lane 5 show distinctive three bands which is due to the topoisomers of
the uncut plasmid . The different migration speed of plasmid ( supercoiled >linear> relaxed )
causing three different bands to be observed. For lane 2, 3, 4, there are some extra bands ligther
than 3kb band are observed due to the star activities of prolonged incubation time during the
experiement . For lane 7, an additional 8kb band is observed due to imcomplete digestion of the
plasmid from slightly higher concentration of enzyme added during experiment. For lane 4,
although both NcoI and SalI restriction enzyme are added to the plasmid of blue colony , but
there are still only one 3 kb band (vector plasmid) observed. This is due to the close proximity in
the cut side between NcoI and SalI without insertion ,producing very small insignificant
fragment size ( 45bp) compared to 3kb. As for lane 8 , there are 3kb band and 0.5kb band
observed , in which the 0.5 kb fragment is the inserted DNA product.

Conclusion:

The inserted DNA product ( beta mouse actin ) is successfully isolated and verified throught the
identification of 0.5 kb bands from the plasmid digestion image. The recombinant DNA is
transformed into E. coli strain DH5 α. Hence, the transformed E.coli cell can be further cultured
and lysed to produce many copies of purified DNA plasmid of beta mouse actin for other future
application.

References:

Frey .M, Rall . S, Roth. A, Hemleben . V. ( 1980) . Evidence for uptake of plasmid DNA into
intact plants ( Lemna perpusilla ) proved by an E.coli transformation assay . Z Naturforsch ,
1980 . 35( 11-12) , 1104-6.

								
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