质粒提取与酶切电泳实验报告.doc

7
分子实验报告 2013030020华天瑞
Preparation of Plasmid DNA, Restriction Enzyme Digestion, and
Agarose Gel Electrophoresis
2014/10/14-21
1 Intro
1.1 Objective
To learn
• The characteristics of plasmid DNA
• The method of plasmid DNA mini-preparation by alkaline lysis and the measurement of DNA concentration by spectrophotometer
• The characteristics of restriction endonuclease
• How to use agarose gel electrophoresis to separate DNAs
To understand:
The principles of purification and quantification of plasmid DNA
1.2 Principle
1.2.1 Plasmid and Vector
Plasmid is a small, independently replicating, piece of extrachromosomal cytoplasmic DNA( double stranded and usually circular ) that is capable of autonomous replication and can be transferred from one organism to another.
Vector serve as carriers to allow replication of recombinant DNA in the host cell, usually a vector covers
• Antibiotic resistance gene: such as ampicillin resistant gene, kanamycine resistant gene, and etc.
• Origin of replication (ori ).
• Multiple cloning site (MSC) or polylinker
• Marker genes: such as LacZ gene.
1.2.2 Alkaline Lysis ( 0.2molNaOH + 1%SDS )
SDS is a kind of anionic detergent. It can break bacterial cells and denature proteins. When bacterial cell wall is broken, the plasmid DNA and genomic DNA will be released out and be denatured in alkali environment. When the solution is neutralized by acidic reagent (such as KAc) , the plasmid DNA will be renatured rapidly due to its smaller size. After centrifugation, the plasmid DNA will be in supernatant, while the genomic DNA will stay in the sediment at the bottom of the tubes together with other cell debris.
1.2.3 DNA Concentration Measurement
Based on the strong absorbance of base pairs (A-T, G-C) at 260nm UV, the concentration of DNA can be measured by spectrophotometry. When detected under neutral condition, A260 is used to calculate the nucleic acid concentration where as the ratio of A260/A280 can be used to estimate the purity of nucleic acid (1.8 for pure DNA).
1.2.4 Restriction Endonuclease
TypeII RE cuts dsDNA at specific restriction sites on specific sequence, producing restriction fragments.
1.2.5 Gel Electrophoresis
Solidified agarose solution has certain size of small pores of which the size is decided by concentration. In the electric field and buffer in neutral pH, negatively charged nucleic acid will migrate toward the positive pole. DNA fragments can be separated by different mobility in gel electrophoresis.
1.2.6 EB ( Ethidium bromide )
EB can bind with DNA through inserting into the base pairs of DNA molecule. Excited by UV, the DNA bands in gel electrophoresis will emit red fluorescence which can be detected easily. The minimal DNA quantity that can be tested by this method is about 10ng.

2 Materials and Reagents
• E.coli DH5 harboring pCMV-Myc-T10(SIPAR)
• TIANprep Mini Plasmid Kit
P1: (1％Glucose, 50mM/L EDTA pH8.0, 25mM/L Tris-HCl pH8.0)
P2: 0.2 mM/L NaOH, 1％ SDS
P3: 5 mol/L Kac, pH4.8
• plasmid pCMV-Myc-SIPAR
• NEB 1kb DNA Ladder
• EcoRI, XhoI（Takara）
• 10×H buffer
• agarose
• TBE/TAE buffer(1×)
• EB (10mg/ml)
• Loading buffer(3×):
0.25% Bromophenol blue
40％(W/V) sucrose or 30％glycerol
3 Procedures
3.1 Preparation of Plasmid DNA
a. Add 500μl Buffer BL to spin column CP3. Centrifuge for 1 min at 12,000 rpm in a table-top microcentrifuge. Discard the flow-throw, and place Spin Column CP3 into the collection tube.
b. Harvest 1.4 ml bacterial cells in a microcentifuge tube by centrifuge for 1 min at 12,000 rpm for 1 min at 20℃, then remove all the traces of supernatant. Then redo with 1.4ml bacterial cells in another microcentifuge tube.
c. Resuspend pelleted bacterial cells in 250μl Buffer P1
d. Add 250μl Buffer P2 and mix thoroughly by inverting the tube 6-8 times
e. Add 350μl Buffer P3 and mix immediately and thoroughly by inverting the tube 6-8 times
f. Centrifuge for 10min at 12,000 rpm
g. Apply the supernatants to the Spin Column CP3, centrifuge for 1min at 12,000 rpm
h. Wash the Spin Column CP3 by adding 700μl Buffer PW and centrifuge for 1min at 12,000 rpm. Discard the flow-through, wash again with 500μl Buffer PW and centrifuge for 1min at 12,000 rpm.
i. Discard the flow-through and centrifuge for 2min at 12,000 rpm
j. Place the Spin Column CP3 in a clean 1.5ml microcentrifuge tube. Add 50μl EB, let stand for 4min, and centrifuge for 2 min at 12,000 rpm.
3.2 Restriction Enzyme Digestion and Agarose Gel Electrophoresis
a. Enzyme digestion of plasmid DNA（pCMV-Myc-SIPAR）
Table 1

Plamid(ng)
Buffer(μl)*
EcoR1(μl)
Xho1(μl)
H2O(μl)
Total volumn(μl)
Ⅰ
201
2
0

0
16.5
20
Ⅱ
201
2
0.5
0
16.0
20
Ⅲ
201
2
0.5
0.5
15.5
20
Digestion at 37 °C water bath for 1 hour.
Add 10 μl 3x loading buffer to each tube, load 15 μl sample for gel electrophoresis.
b. Add 0.8 g agarose and 100 ml 1X TAEinto a flask, microwave agarose melts
c. Insert comb into the mold. Position the comb 0.5-1.0 mm above the plate
d. Pour the warm agarose solution(65℃) into the mold, avoid air bubbles
e. Solify the gel under room temperature for 45 min, then carefully remove the comb
f. Place the gel into electrophoresis chamber full with 0.5×TAE/TBE
g. Load sample 15μl mixture with disposable micropipette. Change the micropipette every time. Add 4 μl 1 kb DNA ladder (50ng/μl) as reference.
h. Electrophoresis at 100V, stop electrophoresis when the band of bromophenol blue is of 4 strings away from bottom of the gel
i. Place the gel into EB working solution (0.5 g/ml) to stain the gel for 3min
j. Observation and photography

4 Results and Discussions
4.1 Spectrophotometry of DNA extraction
Table 2 : Spectrophotometry results of DNA extraction
A260
A280
A260/A280
DNA concentration
0.405
0.214
1.904
201 ng/μl

1
2
3
Ladder
6.0
3.0
2.0
4.0
Generally the ratio A260/A280 of pure DNA is 1.8, smaller than the result. Meanwhile, the ratio of A260/A230 is relatively high(2.783), suggesting that the amount of RNA is small. Analysing by synthesis these two facts, the extraction of DNA may contain certain amount of oligonucleotides which can cause A260/A280 to be higher than reference level.
4.2 Photograph of stained gel
After exposure under UV light, photograph was taken and is shown below.
Well 1 contains untreated plasmid, 2 bands are present on the lane. The fastest and brightest band locates at the length of around 3.0-4.0 kb. Because of the fact that plasmid DNA in supercoiled form moves faster in electrophoresis, bright band at 3-4 kb indicates that most of plasmids collected are in their natural supercoiled form. The other dimmer band at around 6 kb suggests that other conformation of the plasmid DNA also appears in the extraction( DNA with open strand ). Usually, the dimmer band is brighter than shown in our case. One possible explanation is that in procedure c of 3.1, pelleted bacterial cells were not resuspended sufficiently, causing a lose of open strand( as well as some supercoiled form DNA). This can also exlpain the relatively low concentration of DNA(201ng/μl).
Well 2 contains the sample digested by EcoR I alone and single band with the size of approx. 6 kb is presented. Note that total length of recombined pMCV-Myc-SIPAR is 5.7 kb, the observation of single band near 6 kb suggests a total digestion, which match the expectation of a single incision site.
Well 3 contains sample digested by EcoR I and Xho I. Lane 3 has two bands with length slightly shorter than 4 kb and 2 kb, respectively. The lagging band is brighter than the leading band, which is reasonable since the two bands have the same molecular number.
Ladder Well 200ng DNA was added into ladder well, which gives a total mass of 50ng to 3.0 kb fragment. Brightness of leading band in lane 3 is somewhat equal to that of 3.0 kb, suggesting that the sample added contains approximately (50 + 50 * 2 =) 150ng plasmid DNA (note that theoretical length of leading band DNA is 1.9kb and lagging band is 3.8kb). When this value is doubled (recall that we loaded 15μl out of 30), the experimental mass of plasmid (around 300ng) is, more or less, close to the DNA that was added to the mixture( 1.5μl * 201 ng/μl = 302ng ).

Reference
A. Files of experimental outlines provided by teacher on online.
B.

Questions
1. Difference between preparation of plasmid and genome DNA.
Plasmids are small, supercoiled DNA which can be easily renatured by adjusting pH( such as using alkaline lysis). This make the preparation for plasmid easy. While genome DNA is linear and huge in length and always combined with proteins, the preparation is complicated for protein needs to be degraded and the activity of DNase must be low to avoid the degradation of DNA itself.

2. Analysis of African green monkey small polydisperse circular DNA junction region, clone pDM-r1 by BioEdit
• After installation of BioEdit 7.2.5, run the program and create a new alignment (Ctrl + N).
• Choose Sequence à New Sequence, and paste the sequence of African green monkey small polydisperse circular DNA junction region, clone pDM-r1 from database in GenBank.
• Choose Sequence à Nucleic Acid à Restriction Map, default setting is to detect restriction sites for restriction enzymes that recognize a 6-bp fragment.
Mapping outcome is listed below:

BioEdit version 7.2.5 (12/11/2013) Restriction Mapping Utility

(c)1998, Tom Hall
Restriction Map
2014/10/23 1:40:55
100 base pairs
Translations: none

Restriction Enzyme Map:

1 AAAGCTTATCCACCCATGATCAAGTGGGCTTTATCCCTGGGATGCAAGGCTCCAGAATTTCATATTCAGCCAAACTAAGT 80
1 TTTCGAATAGGTGGGTACTAGTTCACCCGAAATAGGGACCCTACGTTCCGAGGTCTTAAAGTATAAGTCGGTTTGATTCA 80
HindIII BclI TaqII BpmI BstF5I ApoI TspDTI
SfaNI TspDTI
Eco57MI NlaIV
BsaJI Hpy188III
BsaJI FokI

81 TTCATAAGTGAAGGAGAAAT 100
81 AAGTATTCACTTCCTCTTTA 100

Restriction table:

Enzyme Recognition frequency Positions
__________________________________________________________________________
ApoI r'AATT_y 1 56
BclI T'GATC_A 1 18
BpmI CTGGAGnnnnnnnnnnnnnn_nn' 1 36
BsaJI C'CnnG_G 2 36, 37
BstF5I GGATG_nn' 1 47
Eco57MI CTGrAGnnnnnnnnnnnnnn_nn' 1 36
FokI GGATGnnnnnnnnn'nnnn_ 1 54
HindIII A'AGCT_T 1 3
Hpy188III TC'nn_GA 1 53
NlaIV GGn'nCC 1 51
SfaNI GCATCnnnnn'nnnn_ 1 32
TaqII CACCCAnnnnnnnnn_nn' 1 28
TspDTI ATGAAnnnnnnnnn_nn' 2 50, 72

Enzymes that cut five or fewer times

Enzyme Recognition frequency Positions
__________________________________________________________________________
ApoI r'AATT_y 1 56
BclI T'GATC_A 1 18
BpmI CTGGAGnnnnnnnnnnnnnn_nn' 1 36
BsaJI C'CnnG_G 2 36, 37
BstF5I GGATG_nn' 1 47
Eco57MI CTGrAGnnnnnnnnnnnnnn_nn' 1 36
FokI GGATGnnnnnnnnn'nnnn_ 1 54
HindIII A'AGCT_T 1 3
Hpy188III TC'nn_GA 1 53
NlaIV GGn'nCC 1 51
SfaNI GCATCnnnnn'nnnn_ 1 32
TaqII CACCCAnnnnnnnnn_nn' 1 28
TspDTI ATGAAnnnnnnnnn_nn' 2 50, 72

Position Enzyme(s)
__________________________________________________________________________
3 HindIII A'AGCT_T
18 BclI T'GATC_A
28 TaqII CACCCAnnnnnnnnn_nn'
32 SfaNI GCATCnnnnn'nnnn_
36 BpmI CTGGAGnnnnnnnnnnnnnn_nn'
36 Eco57MI CTGrAGnnnnnnnnnnnnnn_nn'
36 BsaJI C'CnnG_G
37 BsaJI C'CnnG_G
47 BstF5I GGATG_nn'
50 TspDTI ATGAAnnnnnnnnn_nn'
51 NlaIV GGn'nCC
53 Hpy188III TC'nn_GA
54 FokI GGATGnnnnnnnnn'nnnn_
56 ApoI r'AATT_y
72 TspDTI ATGAAnnnnnnnnn_nn'

Enzymes that do not cut:
_________________________________________________________
AarI, AatII, AccI, Acc65I, AclI, AfeI, AflII, AflIII, AgeI, AhdI, AleI, AloI, AloI
AlwI, AlwNI, ApaI, ApaLI, AscI, AseI, AsiSI, AvaI, AvrII, BaeI, BaeI, BamHI, BanI
BanII, BbeI, BbsI, BbvI, BbvCI, BceAI, BcgI, BcgI, BciVI, BfrBI, BglI, BglII, BlpI
Bme1580I, BmgBI, BmrI, BmtI, BplI, Bpu10I, BpuEI, BsaI, BsaAI, BsaBI, BsaHI, BsaWI
BsaXI, BsaXI, BseMII, BseRI, BseYI, BsgI, BsiEI, BsiHKAI, BsiWI, BslI, BsmI, BsmAI
BsmBI, BsmFI, Bsp1286I, BspCNI, BspEI, BspHI, BspMI, BsrI, BsrBI, BsrDI, BsrFI
BsrGI, BssHII, BssSI, BstAPI, BstBI, BstEII, BstXI, BstYI, BstZ17I, Bsu36I, BtgI
BtsI, Cac8I, ClaI, DraI, DraIII, DrdI, EaeI, EagI, EarI, EciI, Eco57I, EcoICRI
EcoNI, EcoO109I, EcoRI, EcoRV, FalI, FauI, FseI, FspI, FspAI, HaeII, HgaI, Hin4I
Hin4I, HincII, HpaI, HphI, Hpy8I, HpyF10VI, KasI, KpnI, MboII, MfeI, MluI, MlyI
MmeI, MnlI, MscI, MslI, MspA1I, MwoI, NaeI, NarI, NcoI, NdeI, NgoMIV, NheI, NotI
NruI, NsiI, NspI, PacI, PciI, PflMI, PleI, PmeI, PmlI, PpiI, PpiI, PpuMI, PshAI
PsiI, PspOMI, PsrI, PsrI, PstI, PvuI, PvuII, RsrII, SacI, SacII, SalI, SanDI, SapI
SbfI, ScaI, SexAI, SfcI, SfiI, SfoI, SgrAI, SmaI, SmlI, SnaBI, SpeI, SphI, SrfI
SspI, StuI, StyI, SwaI, TaqII, TatI, TspGWI, TspRI, Tth111I, XbaI, XcmI, XhoI
XmaI, XmnI, ZraI