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th exceptions. Steps 5 and 14 were were combined and placed in ice for 30 min.
omitted from the procedure.. With step 10 Bacteria was heat-shocked for 30 sec. in
column was allowed to sit for 5 min.For step
10 in the procedure the QIAquick column Fig. 1 Calpain
sat for 5min. Optional step 9 was amplification Gel
used.followed and the cColumn was eluted electrophoresis. 0.8%
agarose gel for 1 hour
with 30uL of EB buffer. with 1X TAE buffer.
Calpain-ASD band
Restriction digest/ Restriction
appears at 1.5 kb band.
mapping of insert
Restriction digestion of 30ul purified
calpain-ASD PCR product with 1.2ul XbaI 42C water bath and . pPlaced back on ice
(0.5U/ul) and 0.8ul PstI (0.5U/ul) (NEB) for 2 min. Contents was sShaken in 250ul of
were performed in 4ul of reaction buffer #3 soc.SOC medium for 1 hour at 37C. 50ul
(1x)(NEB), loading dye (1x) 8ul, of X-gal (25.5mg/ulL /ul) was added to agar
supplemented with 4ul of bovine serum plates, spread with glass beads, and placed
albumin (1x) (BSA)(NEB). Contents in incubator. and bBacteria plated on agar
combined and place in 37 C water bath for plates at 20ul and 200ul on LB-AMP plates
1 hour. For restriction mapping 10uL of RE and i. Incubated at 37C for 18hrs.
digested plasmid and 1uL of uncut plasmid,
and 1kb ladder were ran in electrophoreisis
gel for analysis. Samples of single colonies of
transformed E.coli DH5 were placed in 4
Contents stored in -20C freezer. Materials
tubes containing 3ul of LB-AMP. Two
purchased at New England Biolabs.
recombinant calpain-ASD/pUC19 colonies
Ligation/ Transformation (white colonies #1 and #2) from
transformation, one vector only colony (blue
DNA ligation with 15-ul Xbal/Pstl colony) from transformation, and 1
cut calpain-ASD gene was incubated with 1 recombinant colony (white colony) from
ul Xbal/Pstl cut phosphatase-treated pUC19 control plate. Cultures were incubated at
in 2ul 1X T4 DNA ligase reaction buffer 37C in shaker overnight at 225rpm.
(50mM Tris-HCl, pH 7.5, 10 mM MgCl2,
1mM ATP, 10 mM dithiolthreitol), 1ul DNA
ligase enzyme (NEB), 1ul ddH2O.
Reactants were placed in PCR
machineincubated overnight (18hrs) at 16C
and. Then set at 65C for 10 min to heat kill
ligase.
Materials procured from New
England Biolabs. Transformation was
performed with 50ul of E.coli DH5 cells
and , 10ul of recombinant calpain-ASD
pUC19 DNAligation mixture. , CcContents
Brain Research (2017) 1-5 Lauren Earley Page |3
0.5-
Brain Research (2017) 1-5 Lauren Earley Page |4
In order to insert Xbal/Pstl digested from the culture toto allow for testing of
calpain-ASD in to and pUC19 vector a presence of calpain-ASD insert in pUC19
ligase reaction was performed. pUC19 was vector.
treated wih phosphatase. Both vector and
Instructions provided by mini prep
insert were combined with 1X ligase buffer
kit were used and plasmid was successful
and DNA ligase enzyme treated with
purified and isolated.
phosphatase and placed in PCR machine
overnight. Ligated calpain-ASD/pUC19 White Blue
weremixture was used to transform E. coli Plate # 1 1White 15
Blue Formatted Table
DH4 alpha bacterial cells. This would allow Plate
Plate #2
#1 51 116
15
for bacteria to take up the recombinant
Positive
Plate #2 336
5 0116
plasmid and replicate it in order to clone our # Control Abs Abs 260nm/280nm Formatted Table
calpain-ASD gene. E. Coli and recombinant Positive 260nm 336 280nm 0 Ratio
plasmid were combined then placed on ice. 1 Control 0.023 0.026 0.885
Then were placed in soc. medium and 2 0.059 0.049 1.204
shaken for 1 hour. X-gal was added to LB- 3 0.055 0.043 1.279
AMP plates to determine if recombinante 4 0.015 0.013 1.154
plasmid was present.and bacteria was plated Purified cloned recombinant calpain-
and incubated. Colonies were produced as ASD plasmid was analyzed to verify the
shown in (Table 1). Plate 1 shows one white calpain-ASD gene was inserted into the
colony and plate two shows 5 white pUC19 vector.Plasmid was analyzed by
colonies. This indicates that there were that spectrophotometer A Spectrophotometer
at least six colonies were an insert is present was used to determine the 260nm/280 ratio
in thewith recombinant plasmid. and the concentration to verify insert was
presentdetermine purity of DNA. The
results were as presented in Table 2. White
Table 1. Colony Counts of bacteria colonies #1 and #2 were tested along with
transformed with ligation mixture recombinant calpain- Blue colony #2 and a white colony for a
ASD, number of colonies found on each plate. White
colonies indicate that an insert is present in the plasmid. positive control.
Blue colonies indicate no insert in the plasmid.
Spectrophotometer results from each colony sample. This Fig. 3 and Fig. 4 Gel
includes two colony samples from our cloned plasmids and electrophoresis of PCR
a negative and positive control. 1-white colony, 2-white product of purified
colony #2, 3-Blue colony #2, 4-Positive control. product to verify
# Abs Abs 260nm/280nm calpain-ASD was
cloned. White colony
260nm 280nm Ratio (and Xbal/Pstl digest
1 0.023 0.026 0.885 recombinant genomic
2 0.059 0.049 1.204 DNA isolated from
3 0.055 0.043 1.279 recombinant calpain-
ASD gene and pUC19.
4 0.015 0.013 1.154 WC#1) and white
colony #2 (WC#2), white colonies taken from bacteria
# Abs 260nm Concentration culture of recombinant calpain-ASD/pUC19. Blue colony Formatted Table
1 0.023 115.0mg/ml (BC), vector with no insert, negative control. Positive
2 0.059 295.0mg/ml control (from bacteria culture as negative control. PC),
white colony with verified insert, positive control. ,
3 0.055 275.0mg/ml positive control. 1kb, 1 kb ladder1 kb ladder (1kb). Run in
4 0.015 75.0mg/ml 1% agarose gel for 45 min. at 100 volts with 1X TAE
All 260nm/280nm ratios are below 1.8 buffer.
which we can conclude that we do not have
pure DNA. Implies significant
contamination in the sample.
Concentrations are present in Table 3.
-8.0kb Table 3. 1:100 dilution, final concentration of the Formatted: Font: 9 pt
-4.0kb plasmid samples. 1-white colony, 2-white colony #2,
-3.0kb
3-Blue colony #2, 4-Positive control.
-2.0kb
# Abs 260nm Concentration
1 0.023 115.0mg/ml
2 0.059 295.0mg/ml
3 0.055 275.0mg/ml
4 0.015 75.0mg/ml
2.0- R.E.Digest
1kb WC#2 WC#1 BC PC
1.5-
1.0-
PCR
1kb WC#2 WC#1 BC PC
R.E. Digest Verification of Insert
Brain Research (2017) 1-5 Lauren Earley Page |7
being unreadable.
PCR
To further verify the presence of the calpain-
1kb WC#2 WC#1 BC PC
ASD sequence in the pUC19 vector 3ul of
sample were sent out to be sequenced.
Forward and reverse sequencing was
compared to Blast data base. Calpain-ASD
did not return as a result of the sequence.
When compared with other vectors and
pUC19 a 97% alignment with expression
vector pFerX8 (fig. 5).
To further verify the presence of the
calpain-ASD sequence in the pUC19 vector
3ul of sample were sent out to be sequenced.
To determine whether the isolated calpain- Forward and reverse sequencing was
compared to Blast data base. Calpain-ASD
ASD insert was properly ligated with did not return as a result of the sequence.
When compared with other vectors and
pUC19 vector and successfully cloned, PCR pUC19 a 97% alignment result returned (fig.
5&6).
and Xbal/Pstl digestion were performed.
Figures 3&4 depict electrophoresis gels to
verify the presence of the calpain-ASD We can conclude that the calpain-
insert in the pUC19 vector. Purified PCR ASD gene was not cloned and is not present
products and R.E. digest products were in the pUC19 vector. Most likely we just
used. All PCR samples including negative have an empty vector.
control (BC) exhibit a band at 1.5kb. This
eludes to contamination within the sample.
The initial calpain-ASD insert 1,689bp, Discussion
therefore the negative control (BC) should
not have a band at 1.5kb if the insert is not Overall our early electrophoresis gels
present. Xbal/Pstl digestionR.E digest show that we did amplify, isolate and purify
resulted in white colony 2 (WC#2) the calpain-ASD gene. However, we were
exhibiting a band between 1.5kb, 2.0kb, and not successful in transforming a bacterium
3.0kb. White Colony one (WC#1) was void with the recombinant calpain-ASD/pUC19
of any bands. Blue colony (BC), the plasmid. We were not able to successfully
negative control, shows a dark band at 3.0kb clone the calpain-ASD gene. Results eluded
and faint bands at 4.0kb and 8.0kb. Uncut a lot toto significant contamination in the
plasmid was originally included in the gel sample. We were successful in PCR to gain
Brain Research (2017) 1-5 Lauren Earley Page |8