rtpcr(rt pcr)
山东高考专科分数线-元宵节祝福语
rt-pcr（rt - pcr）
I. Experimental
apparatus and materials:
1, transfer
guns: 1ml, 200 L, 20 L, 10 L, 2 L
2,
suction head: 1ml, 200 L, 20 L
3,
homogenate tube: 5ml
4, suction head
table: put 1ml suction head one, put 20 l suction
head one
5, EP tubes: 1.5ml, 0.2ml,
100 L
6, reagent bottle: 2 60ml Brown
reagent bottle (wide mouth, with
cover)
1 125ml white reagent bottles (with absolute
ethanol)
7, 50ml, 250ml, 500ml: a
8, capacity bottle: 250ml, 500ml, 1000ml
9, test tube rack: 5ml, 1.5ml, 20 L
10, salt water bottles: 250ml, 500ml each 2
spare, one with
absolute ethanol, another with
DEPC water
11, aluminum lunch box: 4
12, plastic small lunch box: 1
13, large porcelain cylinder: 2
14,
tin paper: a roll
15 rolls of paper: 2
rolls
16, triangle flask: with a cap,
slightly larger
Two, the processing and
preparation of experimental equipment
1,
plastic products: (including gun head, EP tube,
homogenate
tube, etc.)
The DEPC of
water from the flask into the ceramic cylinder,
the
plastic products by soaking them, which
requires a small tip
Straw DEPC into the
water, and then dried overnight, high
pressure, spare, before the experiment will
first put the gun
suction head, and a high
pressure (EP tube)
2, glass products:
acid soaking overnight, washed clean, dry
spare foil Mongolia (DEPC blister) (wash after
the first bubble
1 per thousand DEPC
overnight, and then dried)
3,
homogenizer: (including scissors, tweezers) first
wash, and
then high pressure (do not need to
bubble DEPC)
Three. Reagent preparation:
1, DEPC water: suck 1ml, placed in 1000ml
double steamed water,
with 1 per thousand DEPC
water, placed in the 1000ml capacity
bottle, static 4 hours standby.
2, 75% ethanol: with anhydrous ethanol DEPC
water, and then put
-20 degrees preservation
(where DEPC water should be high
pressure)
3, isopropyl alcohol: put brown bottle
4, chloroform: put in brown bottle
5 agarose
Four. Preparation of
several buffers:
1 Electrophoresis
buffer:
Tris 54G
Boric acid
27.5g
0.5M, EDTA, 20ml? PH8.0
Distilled water 1000ml
5 x TBE (Zhu
Cunye)
The 5 x TBE is diluted 10 times to
0.5TBE, which can be used
in electrophoresis
(i.e., the concentration of the working
fluid), such as 50ml storage solution, 450ml
water, 500ml
working buffer
2,
the sample buffer:
0.25% bromo phenol
blue
0.25% xylene green FF
30%
glycerin
6 * buffer solution, stored at 4
DEG C
Five. Preparation of agarose gel:
1 and 1%:
1.0g 100ml agarose
electrophoresis buffer, microwave oven fire
30
seconds to boiling, adding 2.5 L agar 10mgml
ethidium
bromide cooled to 60 DEG C melting,
mixing, will warm into the
gel film has been
set a good comb, placing 30-45min at room
temperature after the electrophoresis.
2 and 1.5%:
Ditto, change the amount
of agarose to 1.5g
Six, the purchase of
Rt-PCR materials:
(working man. Tel.
2236106.)
Taq enzyme (containing MgCl2,
Buffer) 200u 70
DNTP: 1
Oligo (dT) 15: 1 OD 40 Promega
M-MLV:
1, 250, Promega (Buffer)
RNasin: 1, 110
---20 DEG C
DEPC 5g 110
Trizol 100ml1 bottles Invitrogen Life
technologies
- 4 degrees centigrade
Marker: 10 g 0.2 gml 150 Kuwait
(1543., 994., 697., 515., 377.231)
Seven. Primer synthesis
Justice: 5
'-CACGATGGAGGGGCCGGACTCATC-3'
Antisense:
5 '-TAAAGACCTCTATGCCAACACAGT-3'
2, par-4:
Justice: 5 '-GGGACCTCGGAACTCAAC-3'
Antisense: 5 '-TGTATCTGCCTGGGACTG-3'
3. Calculation of annealing temperature
2 (A, T) 4 (G, C)
The average
number of positive and negative averages
fluctuates
again and again (+ 4 DEG C, or + 5
DEG C)
4. The primers are 5 OD each, and
each OD bottle is packed
separately
5
primer dilution:
The amount of water
added to DEPC is (L)
= nmol OD * on the
tube marked OD * 100
Is a primer solution
for 10p mol L concentration
Eight and
PCR electrophoresis
The first 1-10 L PCR
products, and has little on paper by LAN,
repeatedly playing suction after mixing by
electrophoresis,
the general current of 10mA,
power 100V, electrophoresis 30
minutes,
observed under ultraviolet light, satisfactory
results for scanning and printing.
Nine, several points of attention:
1,
the key step of reverse transcription is immediate
ice water
bath
2, Rt, first
open PCR preheat 30 minutes.
3, RNA
pumping ahead, open the centrifuge pre cooling.
Total RNA was extracted by TRIzol
Cells 1 * 107
Tissue 100mg
Here
Plus 1mlTRIzol
The
cells were blown to the liquid with a 1ml sampler
and
clarified without cell mass
Here
Homogenate (thoroughly, then transferred
to the EP tube)
(tissue homogenate >100mg
1ml EP tube each time)
Here
Mix
it upside down 10 times, room temperature for 5
minutes
Here
Chlorination of 15
volume (0.2ml) (must press the total volume
of
15)
Here
Mix it upside
down 10 times, room temperature for 5 minutes
Here
4 DEG C, centrifuged 12000g, 15
minutes
Here
Transfer to the
upper aqueous phase (about 400 l) in another
1.5mlEP tube
Here
Add an
equal volume of isopropyl alcohol (about 400 l)
and mix
it for 10 minutes at room temperature
Here
4 DEG C, centrifuged
12000g, 10 minutes
Here
Supernatant
Here
75% ethanol
(DEPC water) 1ml with ice
Here
Centrifuge at 7500g for 4 minutes for 5
minutes
Here
Discard the
supernatant and let the air dry for 5-10 minutes
(not completely dry)
Here
Soluble in DEPC water to 20 l (10 L-20 L)
(water soluble at <10 for 55-60 minutes.)
Note:
1, RNA if used for nucleic acid
transfer, should be dissolved
in the sample
buffer, or DEPC dissolved
2, the cell
tissue plus TRIzol homogenate can be placed at
least
-60 months (or more than a year)
3, RNA in 75% ethanol, 2-8 DEG C can be stored
for at least one
week, -20 DEG C can be stored
for at least one year
Two step RT-PCR
(step 1: reverse transcription)
Concentration of reagent
RNA
23 Mu L (11.5 L)
Oligo (dT) 150.05 mu g
Mu L, 4 Mu L (2 L) 0.005 mu g Mu L
(diluted 10 times)
Here
Mixing, centrifugation, 70 5min
Here
Immediately ice bath, slightly
centrifugal
Here
Concentration
of reagent
M-MLV Buffer 5 * 8 L (4 L) 1 *
DNTP 10mM 2 mu L (1 L) 0.5mM
RNasin 40U Mu L 1 mu L (0.5 L) 20 mu
M-MLV 200U Mu L 2 mu L (1 L) 200U
The
total volume is 40 L (20 L)
Here
Mixing, centrifugation, 42 60min
Here
95 10min (destroy MLV)
Here
4 degrees preservation
Two step RT-PCR
(second step: PCR
reaction)
The total volume is 20 l (50 L)
Concentration of reagent
Taq
Buffer 10 * 2 L (5 l_) 1 *
MgCl2 25mM 1.2
Mu L (3 L) 1.5mM
DNTP 10mM 0.2 Mu L (0.5
L) <200uM
Upstream primers 10pmol, Mu L,
0.3 Mu L (1 L) 10pmol
Downstream primers
10pmol, Mu L, 0.3 Mu L (1 L) 10pmol
CDNA
template X () (1-10 L)
DEPC water 20
l-4.3 l-X
Taq enzyme 2.5U Mu L,
0.3 Mu L (1 L) 2.5U Mu L
Here
Mix
Here
97 degrees, 5
minutes
Here
Instant ice bath
Here
Mix
Here
Pre denaturation at 95, 5
94 DEG C,
30 second degeneration
Annealing at 40 X
72 degrees, 30 seconds extension
72 point, 7 point, end extension
28-36 cycle, 4 degree heat preservation
Three electrophoresis:
Add 1-10
Mu L PCR product bromine Finland (1-2.5 L) mix,
add
sample, electrophoresis.
Note:
Taq enzymes, M-MLV and Rnasin are stored at -20
DEG C and
placed on ice during operation. DNTP
do not freeze again and
again.