Protocol

1.Extract Arabidopsis genomic DNA

Material:

EZ-10 Spin Column Plant Genomic DNA Purification Kit

β-mercaptoethanol

RNaseA

Chloroform

Ice box and ice

2.PCR

Material:

ddH20

dNTPs mix

10×Buffer

Taq polymerase

PCR Primers

Arabidopsis genomic DNA

Methods:

add material into a 0.2ml EP tube according to the table

Material	dosage/μl
dNTPs mix	1
Forward primer	0.5
Reverse primer	0.5
Arabidopsis genomic DNA	3
ddH20	12
Taq polymerase	0.5
Total	20

set the PCR program

For promoter cop1,phyB,pif1,gene hhl1,gene flu:

94℃ × 5min +(94℃×30s +Tm×30s +72℃×1min)×35+72℃×5min +4℃(for preserving)
For pHhl1-gene hhl1:

94℃ × 5min +(94℃×30s +Tm ×30s +72℃×90s) ×30+72℃×5min +4℃(for preserving)

Tm of each primer:

Name	Tm(Forward/Reverse)(℃)	Tm in PCR(℃)
Promoter pif1	62.59/62.67	63
Promoter cop1	66.77/65.46	66
Promoter phyB	63.94/63.90	64
pHhl1-genehhl1	60.75/59.38	60
gene hhl1	60.90/59.38	60
Fluorescent gene flu	62.42/66.90	65

3.digestion

Material:

ddH20

BsaⅠBuffer

PstⅠBuffer

Restriction enzyme BsaⅠ，PstⅠ，EcorⅠ

PstⅠBuffer:100mM NaCl、50mMTris-Hcl、10mM MgCl2、100μg/mlBSA、ph7.9

Bsa1 Buffer:50mM CH3COONa、20mMTris-Hcl、10mM(CH3COO)2Mg、100μg/mlBSA、ph7.9

Method

add materials into a 1.5ml EP tube according to the table below:

Material	dosage/μl
PCR product(after purification)	17
restriction enzyme buffer	3 (/4)
restriction enzyme	1(0.5 each)
ddH20	4
Total	25

PCR product	cutting sites	restriction enzyme Buffer
pHhl1-hhl1	BsaⅠ+EcorⅠ	3μlPstⅠ+1μlBsaⅠ
hhl1	PstⅠ+EcorⅠ	3μlPstⅠ
flu	EcorⅠ+BsaⅠ	3μlPstⅠ+1μlBsa1Ⅰ
cop1	BsaⅠ+PstⅠ	3μlPstⅠ
phyB	BsaⅠ+PstⅠ	3μlPstⅠ
pif1	BsaⅠ+PstⅠ	3μlPstⅠ

Place the tube in a 37℃ incubator, stand for 1 hour.

Water bath the tube in 65℃ for 20 mins to end the digestion reaction.

4.chemical conversion

(1)Preparation of LB Broth

2g peptone

1g NaCL

0.2g glucose

1g yeast

Add water to 200ml, regulate the pH at a range of 6.8~7.2

(2)Preparation of LB Broth Medium

(3)Preparation of chloromycetin solution

10ml ddH20

0.25g Chloromycetin

Add trace amount of NaOH until the chloromycetin dissolves completely

(4)Add chloromycetin solution into LB broth, mix them

(5)Add competence DH5α （100μl）into 10 μl plasmid，mix them gently, and ice bath

(6)Heat the solution in 42℃ water bath，and ice-bath 2min (Be sure not to shake the centrifuge tube)

(7)Add 890μl, 37℃ preheating LB broth, mix them gently ,shake them in the condition 37℃, 150rpm for 45min.

(8)4000rpm centrifuge 5min, discard 900 μl supernatant ,suspend the 100μl sediment gentlely

(9)Spread the solution on the LB broth medium evenly, after it is dried, culture it upside down in the 37 ℃ incubator for 16 hours

(10)Put the centrifuge tube which including culture solution and competence DH5α in the bed temperature incubator, shaking culture it in the condition of 220rpm, 37℃ for 16 hours

*THE RESUALT：

(1)The escherichia coli didn't grow on the LB broth medium

(2)After centrifuged, some escherichia colis can be found with a little amount in the bottom of the centrifuge tubes

5.plasmid extraction

Experiment macterial: the kit for plasmid extraction

Experiment apparatus: centrifugal machine, water bath, incubator

Experiment preparation: 60 ℃ water bath， 37 ℃ incubator

(1)centrifuge the bacterial liqiud ,12000rpm 1min. Discard the supernatant.

(2)According to the kit for extraction, extract the plasmid DNA

(3)Preserve the plasmid in 4℃ refrigerator

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6.extraction of RNA

Experiment macterial：the kit for RNA fractionation and purification

Experiment steps: refer to protocol

7.RT-PCR

Experiment macterial：M-MuLV

(1)Preparation for RT-PCR

Buffer 5μl

Forward Primer 2μl

Reverse primer 2μl

RT reverse transcription 0.5μl

Taq enzyme 0.5μl

RNA 20μl

ddH2O 19.5μl

(2)RT-PCR program

45℃×30min+94℃×5min+（94℃×30s+60.5℃×30s+72℃×1min）×40+72℃×10min

GDSYZX-United/NOTEBOOK/protocol

Protocol