105S3 BIOTECHNOLOGY CORE TECHNIQUES

DNA experiment report

Abstract
This report is for 105S3 BTC course experiments of DNA parts. The DNA parts
composed by 6 sections, conducted by sequence from DNA-1 to DNA-6. The contents
of these experiments are basic DNA cloning skills and micropipetmen usage and
practice. This report includes the documentation of experiment results, the material
and method we used and the discussion based on what we did and observed.
Introduction
Cloning is the most basic skills of all molecular biological experiment. Its almost
impossible for a biology related researcher not to do it, so how to do it properly and
stably is very important. The main purpose of the DNA part of BCT experiment is to
construct a plasmid which has a gusA gene, cut and isolated from plasmid pBlueGus,
cloned on pQe31 Vector. This whole process involves several basic cloning techniques
including bacteria culture incubation, mini-prep of plasmid, , , , and .
GusA is a gene encoding -glucuronidasean, a enzyme from the bacterium
Escherichia coli, which can turn some kind of colorless glucuronides substrate into
detectable coloured or fluorescent compounds. This character of gus gene is widely
used as a kind of reporter system in the molecular biology field to visualize or
quantify the insertion of target sequence or the expression ability of promoters. The
most used substrates for gus reporter systems include X-gluc (5-bromo-4-chloro-3-
indolyl glucuronide), p-nitrophenyl -D-glucuronide and MUG (4-methylumbelliferyl-
beta-D-glucuronide).1
pQe31 is an expression vector which has T5 promoter to express the genes
inserted into its MCS. 5 promoter originally came from bacterial phage T5 early
promoter, but it requires no phage polymerase to work compared to T7 promoter.2

Material and methods

1. Introduction and cloning tools practice
Examination of the accuracy of the micropipetman We set P100, P200, P20
on its maximum volume, and then used it to take and weight distilled water.
This process was repeated 5 times for each pipetman and the results were
recorded. Set up RE digestion pBlueGus was digested by BamHI, PvuII, ScaI
together and separately with 10XNEBuffer. For each reaction, total Volume
 was 20L. After completion of the RE digestion, reactions were stopped by 65
water bath for 15min. Electrophoresis The digested DNA samples were
used to conduct the electrophoresis analysis in 1.2% agarose gel with 0.1%
EtBr added. The electrophoresis was run at a constant voltage of 50V or 100V.
Digestion of plasmid DNA(2.2.1) Plasmid pBluescript, pQe31 and pBlueGus
were digested by HindIII and BamHI. Due to their different pH condition
requirements, we conduct the two RE digestion one by one. After the RE
digestion, an electrophoresis analysis was done to check if it indicates good
quantity and quality or not.
2. Culture method for bacteria
Isolation of a single bacterial colony To produce single bacterial colony, we
streaked bacterial colony on the LB agar plate with a sterile and cooled down
inoculation loop. overnight suspension culture A JM109 single bacterial
colony was scraped up and put into 2 mL LB broth, and the medium was
incubated at 37 overnight.
3. Mini-Preparation of plasmid DNA ( 2.1)
After overnight 37 incubation, we collected bacteria cells by centrifuge.
Cells then were resuspended and lysed to release DNA. Chromosomal DNA
were separated and remove by MPI,II,III solution process, and plasmid were
collected by column, finally dissolved in TE-8.0 solution.
4. Purification of DNA fragments
Set up large scale digestion ( 2.2.2 step 1) (2.2.2 step 2) Plasmids prepared in
mini-preparation were digested by Restriction enzymes HindIII and BamHI at
37 with 10X buffer B overnight. A little part of reaction was used to
examine the completion of digestion by gel electrophoresis. Silica gel
absorption The digested plasmids electrophoresed on a 1% preparative
agarose gel containing EtBr, and the wanted bands, gusA and pQE31, were cut
out and dissolved in buffer QG. DNA fragments were collected and extracted
by QIAquick spin column with buffer PE and EB, which all from QIAquick Gel
Extraction Kit(QIAGEN).
5. DNA quantification(2.4)
A rough estimation of DNA concentration was conduct by comparing the
brightness of EtBr signal betweenMr. and DNA samples after under UV
exposure after an electrophoresis. The precise measure of DNA concentration
was conducted by TA using nanodrop machine in TechComn.
6. Set up ligation reactions (2.5)
Purified gusA and pQE31 BamHI/HindIII fragments were mixed together with
5X T4 DNA ligase buffer and T4 DNA ligase added, incubated at 12
 overnight.
7. Preparation of competent cells / transformation
Preparation of competent cells 40mL JM109 bacterial culture was repeatedly
centrifuged and re-suspended with 0.1M CaCl2 solution, made final
component cells with volume of 1.6mL. A tube of commercial component
cells from Protech Technology Enterprise was used in transformation directly
for comparison too. Transformation <10L DNA samples and 200L
component cells were added and mix in each tube. Heat shock 42 for 90s
and recover for 60~90min in 800L SOC broth at 37. Cells then were spread
on SOC/Amp plates, incubated at 37 for 12~20hr
8. Colony hybridization 2.7.1 / Histochemical detection 2.7.2
A nitrocellulose membrane was placed on the colonies to dip a little cells of
each colony. To induced gusA gene expression, the membrane was place on
IPTG containing plate. Colony hybridization we use LB/Amp/IPTG plate for
induction. Then cells were lysed to release the proteins they have, and the
proteins were hybrid with the primary(AntiGus antibody) and secondary(Goat
anti-mouse horseradish peroxidase-conjugated antibody) antibodies. Finally,
we use DAB to conduct the coloration. Histochemical detection The
membrane was placed on a LB/Amp/IPTG/X-Gluc plates, so the product of
induced gusA gene could turn X-gluc into colored compound and stain the
membrane with blue color.
9. Quick screening 2.7.3
Several colonies were selected and for this experiment. They were scraped up
and swirl in 30L 1XNET. The solution then was used to re-suspend the pellets
collect by centrifuge from bacterial culture. After collection of cells, we used
PCI and CI to extract, collect and wash DNA samples, then did a 1% agarose
gel electrophoresis at 50V.
10. Southern blot Mini-Prep enzyme digest electrophoresis
After the RE digestion and electrophoresis, the DNA in the gel was transferred
to a Nylon membrane by capillary blotting action for overnight. The
membrane was baked by DNA crosslinker to fix DNA to it.

Results and Discussion

Agarose gel electrophoresis for RE digestion check (fig.1)
Figure 1 : Lane1=1Kb DNA ladder, Lane2~8= pBluescript, pBluescript/BamHI, pBluescript/PvuII,
pBluescript/ScaIII, pBluescript/BamHI/PvuII, pBluescript/BamHI/ScaIII, pBluescript/PvuII/ScaIII

The product of Mini-prep of plasmid pBlueGus and pBluescript were analyzed by

electrophoresis. The result shows at (fig2). We can see every fragments appeared at
predicted position except #4. #1,4,7 are uncut circular plasmid, so these bands
shouldve appeared at the lower position compared to the same size linear plasmids
#2,5,8. Large scale digestion of pBlueGus and pQE31 by HindIII and BamHI was set up
on 7/14. When it completed on 7/15, some of reaction were analyzed by
electrophoresis(fig.3). The result indicated our digestion completed well and
correctly. Another gel electrophoresis was done with basically same set of the 7/14
one, and its result show at (fig.4) in the picture, we can see almost every band in the
formal one, but and the #4 apeared at the lower and correct position.

Figure 2
 3 Figure 3:
lane 1=1kb ladder,
lane2/3=pBlueGus/pQE31 reaction
Figure 4:
Lane 1=1Kb ladder
Figure 5:
Lane 1/2=Mr 1L/2L, lane 3=pQE31,
lane4=gusA
Figure 6:

4 5

The concentration estimation of gel extraction products pQE31 and gusA fragments
is showed at (fig.5). Quick screening electrophoresis result is showed at (fig.6)
Results of 7/20 colony hybridization and histochemical detection are showed at
(fig.7), 7/22 electrophoresis of RE digestion of purified plasmid is showed at (fig.8)
 histochemical detection
7

colony hybridization

Fig.8: lane17= 1Kb DNA ladder, lane1~16=pBlueGus/H/B, pQE31, pQE31/H, pQE31/H/B,

clone1, clone1/H, clone1/H/B, clone2, clone2/H, clone2/H/B, clone3, clone3/H, clone3/H/B,
clone4, clone4/H, clone4/H/B (H=HindIII, B=BamHI)

transformation

Reference

1 https://en.wikipedia.org/wiki/GUS_reporter_system
2 http://parts.igem.org/Part:BBa_K592008