GENERAL MATERIALS AND METHODS
2.1 Nucleic acids related techniques
2.1.1 Mini preparation for DNA
QIAprep® Spin Miniprep Kits (QIAGEN, Cat. No. 27106) was used to purify the
plasmid from E. coli. Simply, 2-3 ml of overnight cultured E.coli in LB medium was centrifuged
and the pellet was collected. The pellet cells were then suspended in 250 µl of Buffer P1. 250 µl
of Buffer P2 was added and the mixture was inverted for 6 times till the mixture become clear.
350 µl of Buffer N3 was then added and the mixture was inverted for 6 times till the solution
became cloudy. The mixture was then centrifuged for 10 minutes at 13,000rpm. The supernatant
was collected and the QIAprep spin column was used and was centrifuged for 1 minute. The
flow-through was discarded. The QIAprep spin column was washed by adding 0.5 Buffer PB and
centrifuge for 1 minute. The flow-through is discarded. The column was further washed by
adding 0.75ml of Buffer PE and was centrifuged for 1 minute. The flow-through was discarded,
and the column was centrifuged for additional one minute. The column was then moved to a new
microcentrifuge tube. 30 µl of water was used to elute DNA by standing for 1 minute and
centrifuging for 1 minute at 13,000 rpm. The eluted DNA was confirmed by either running gel
electrophoresis and/or sending for DNA sequencing.
2.1.2 Midi preparation for DNA
Bacteria carrying DNA plasmid were cultured in 1ml of LB medium supplemented with
antibiotics (50ng/ml Kanamycin or Ampicillin). Then, the bacteria culture was transferred into
150ml warm LB medium and cultured overnight at 37°C to amplify bacteria together with
minutes. 3ml of resuspension buffer was used to suspend the cell pellet, and 3 ml of lysate buffer
was used to lysis the bacteria. The lysate was neutralized by adding 3 ml of neutralization buffer
and inverting the mixture. Then, the solution was centrifuged at 15,000 for 25 minutes and the
supernatant was carefully collected into a 50 ml conical tube. 10 ml of DNA purification resin
provided with the kit was added to the same tube and mixed gently. The mixture was loaded to a
mini preparation column and the vacuum equipment was applied to remove supernatant. The
resin beads bounded with DNA plasmids were washed with washing buffer twice (10ml each
time) by using the vacuum equipment. 300 µl of pre-warmed ddH2O (~70°C) was added into the
dehydrated resin. Then, DNA plasmid was eluted from the column by centrifuging at 13,200 rpm
for 1 minute. The plasmid DNA may be further purified with phenol/Chloroform/Ethanol
method and the concentration of the DNA was measured with spectrometry at OD260nm.
2.1.3 Agarose gel electrophoresis and gel extraction of DNA
A double stranded DNA binding dye, Ethidium Bromide, can be used to visualize
plasmid DNA or DNA fragments under a UV transilluminator. 1% agarose gel was prepared by
adding 1g of agarose into 100ml of TAE buffer. The mixture was heated with microwave to
completely dissolve agarose. The solution was cooled down to 50-60°C with a water bath before
adding 6 µl of ethidium Bromide into it. The solution was then poured into a gel casting
apparatus. Well inserts was set up to make suitable well. 8~10 µl of DNA ladder was loaded into
one well to indicate the molecular weight of DNA fragments. The gel ran at constant voltage of
120V until the DNA/DNA fragment was separated and visualized under the UV transilluminator.
2.1.4 Quantification of DNA or RNA
The concentration of nucleic acids was measured in a spectrophotometer (UV-1700
formula: DNA concentration (µg/ml)=(OD260nm)X(diluted factor)X(50 µg DNA/ml)/(1
OD260nm unit). The purity of DNA was determined by the ratio of the absorbance of
OD260nm/OD280nm. The purity of DNA was acceptable when the ration is between 1.8 and 2.0.
2.1.5 Polymerase chain reaction (PCR) method
PCR method was applied to amplify a target sequence from DNA including plasmid
DNA. PCR method also can be used to mutate specific nucleic acids of a plasmid vector. In
general, 0.5 µg of template DNA was mixed with 1mM of dNTP, 0.5 µg of both forward and
reverse primers, and 1-2 unit of Pfu polymerase in 1X reaction buffer. The reaction mixture was
denatured at 95°C for 2 minutes and then was followed by 25-35 cycles: 95°C denaturation for
30 seconds, 57-62°C annealing for 45 seconds, 72°C elongation for 45seconds (the elongation
time can be adjusted based on the length of the target sequence). An additional elongation step at
72°C for 10 minutes was performed to stabilize the double strands. The PCR product was
examined by agarose gel electrophoresis.
2.1.6 Restriction enzyme digestion and plasmids construction
CDNA of target gene needs to be constructed into the vector in order to exogenously
introduce proteins into the cells. The plasmid needs to be chosen based on the purpose of the
research. The sequences of the vectors are available from the companies. The primers
incorporating the digestion site(s) used for cloning the target gene would be very carefully
designed. The restriction enzymes have optimum conditions therefore the right buffer reaction
system is critical to make the digestion successful. The restriction digestion could be carried by
using one restriction enzyme one time or by using two restriction enzymes one time. In general,
in the final digestion system, 1 µg of the plasmid DNA or 30 µl of PCR product and 1 µl of the
for 2hours or even longer. Agorose gel electrophoresis was used to separate the digested PCR
product or digested vector. T4 DNA ligase was applied to ligase the digested DNA into the
digested vector. The 3’-OH of one nucleic acid and 5’ phosphate of the other nucleic acid can be joined by T4 DNA ligase. The recommended ration of the DNA to plasmid vector was around
3:1. The ligation process was performed in 1X ligation buffer with 1-2 µl of T4 DNA ligase. The
mixture was incubated at 4°C overnight or at room temperature for 30 minutes. The ligation
product containing the ligased vector was transformed into competent bacteria on the second day.
2.1.7 Site-directed mutation method
QuikChange II XL site-directed mutagenesis kit (stratagene) was used to perform a site-
directed mutation. The primer for the mutation can be designed on-line (The Quikchange primer
design program). 0.5 µg of primers was applied to PCR reaction mixture. 2 µl of DpnI enzyme
(20U) was added to the reaction system to digest the parental plasmid vector. The digestion
reaction was performed at 37°C water bath for 2 hours. The mutated plasmid vectors were
transformed into XL-1 Blue competent cells after the DNA was purified. The transformed XL1-
Blue cells were coated on the agar plate containing an appropriate antibiotic overnight at 37°C.
On the second day, several colonies were picked and were cultured in 2ml of LB medium
overnight at 37°C. DNA plasmid was isolated by mini-prep from the bacteria. DNA sequence
technique at the core facility of GSU was used to identify the mutated DNA sequences.
2.1.8 Ethanol purification of DNA
Ethanol purification was performed to remove salt or protein in DNA samples. At first,
DNA solution was added and mixed well with 1/10 volume of 3M sodium acetate. The mixture
was then added and wixed well with 2 volumes of ice-old 100% ethanol. The mixture was
the DNA was washed with ice-cold 70% ethonal twice and centrifuge at 4°C for 5 minutes. The
supernatant was removed and then the DNA pellet was dried by air at room temperature until the
pellet becomes transparent. The concentration of the DNA was determined at OD260nm in
spectrophotometer after the DNA pellet was dissolved in ddH2O.
2.1.9 RNA extraction from tissue
Whole spleens from mice were collected and submerged into 1.5ml of the RNAlater
RNA stabilization reagent (QIAGEN, Cat. 76104). The tumor was cut into small pieces (0.5
cmX0.5cmX0.5cm) and was consequently put into the same amount of the reagent. The samples
were kept at 4°C for overnight and were then moved to -20°C (for less than 4 weeks). 1/3 of the
spleen was collected, and the total mRNA was isolated by the mini-plus kit (including the
shredder column). In detail, the spleen tissue (~30mg) was homogenized in Buffer RLT which
had been added with B-mercaptoethonal. The tissue lysates were then added into the
QIAshredder spin column (QIAGEN, Cat. 79654). Then the lysate were centrifuged for 3
minutes at the maximum speed. The supernatant was transferred into a gDNA Eliminator spin
column and centrifuged for 30s at 10,000rpm. The flow-through in the tube was added with 1
volume of 70% ethanol and mixed by pipetting. The mixture was transferred to an RNeasy spin
column and centrifuged for 30s at 10,000rpm. The flow-through was discarded. 700 µl of Buffer
RW1 was added into the RNeasy spin column and the flow-through was discarded after
centrifuging. 500 µl of Buffer RPE was added into the column. The flow-through was discarded
after centrifuging. And this step was repeated once. The trace amount of Buffer RPE was
removed by centrifuging the column in a new tube for 1 minute at the full speed. The total RNA
was eluted into the collection tube from the column by adding 30-50 µl of H2O into the column
2.1.10 Reverse transcription PCR
The generation of cDNA from mRNA can be performed by reverse Transcription PCR. 1
µg of total RNA extract and 1 µl of oligo dt15 was mixed in a final volume of 5 µl by adding
RNase-free H2O. The mixture was incubated at 25°C for 5 minutes. The mixture was then
incubated at 70°C for 5 minutes. After that, the mixture was incubated on ice for 5 minutes.
Among each reaction, 1 µl of reverse transcriptase, 1 µl of RNase inhibitor, 4 µl of MgCl2
(25mM) and 4 µl of 5X reaction buffer were added together and mixed thoroughly. The mixture
was incubated at 25°C for 5 minutes, 45°C for 1hour and 70°C for 15 minutes. The final RT-
PCR products were then stored at -80°C.