MICB ABI PRISM 310 SEQUENCING GUIDE
SEQUENCING OF PLASMID DNA
Plasmid DNA Preparation
Introduction:
• I have always used the classic “Alkaline Lysis” miniprep method to isolate plasmid DNA. (See below)
• If you prefer miniprep kits, then any commercial plasmid prep kit should be fine. ABI recommends Qiagen purification kits like
the Qiagen QIAprep Spin Miniprep Kit (50) Cat.#27104 for plasmid minipreps. If you are using the Qiagen QIAprep Spin Miniprep Kit ALWAYS BOIL the isolated DNA prior to quantitation. We have noticed that Qiagen Miniprep DNA is sometimes
somewhat "insoluble" and boiling the sample resolves this problem.I have also successfully used Sigma's GenElute Plasmid
Purification MiniPrep Kit, Product Code PLN-10 (10) or PLN-70 (70 purifications). No matter which method you use, remember to dissolve your sample DNA in ddH2O and not TE as EDTA inhibits the ABI sequencing reaction.
• Finally, always use 100% ethanol for precipitations. Impurities in 95% ethanol will inhibit the sequencing reaction.
Alkaline Lysis Method (DD & DT Modifications)
Reagents Needed:
Lysis Buffer
6.25 ml 2M Tris pH8 25mM
10 ml 0.5M EDTA 10mM
50 ml 0.5M Glucose 50mM
433.75 ml double distilled water
Store refridgerated.
NaOH / SDS
4 ml 5M NaOH 0.2M
2 ml 20% SDS 0.2%
94.67 ml double distilled water
(prepare fresh, DO NOT place on ice)
Potassium Acetate Solution
300 ml 5M KAc
57.5 ml Glacial Acetic Acid
142.5 ml double distilled water
Store refridgerated
Phenol:Chloroform:Isoamyl Alcohol (25:24:1) buffer saturated
Sigma-Aldrich Cat# P-3803
Chloroform:Isoamyl Alcohol (24:1)
Sigma-Aldrich Cat# C-0549 or prepare
RNaseA (Ribonuclease A)
Sigma-Aldrich Cat# R-4875
Preamble:
The solutions used in this mini-prep procedure are those of the large scale plasmid prep. In this mini-prep procedure various steps are
combined and shortened to speed up the procedure. This method works very well to OK with most but NOT ALL plasmid / bug
combinations. BEWARE! (ul = microlitre)
Procedure:
• Using a fresh overnight culture of the plasmid containing bacteria, add 1.5 ml of this culture to an eppendorf tube.
• Pellet the bacteria by microcentrifugation for 1 min and then aspirate off the culture media.
• Add 200 ul of the LYSIS BUFFER and resuspend the pellet by vortexing. Make sure that the pellet is totally resuspended.
• Add 200 ul of the NaOH / SDS solution and mix by inverting until the solution becomes clear.
• Add 200 ul of POTASSIUM ACETATE solution and vortex until a white precipitate forms.
• Microcentrifuge for 5 min at top speed (>12,000 rpm). Remove the liquid into a fresh eppendorf tube.
• Add 500 ul of Phenol:Chloroform:Isoamyl Alcohol (25:24:1) and vortex vigorously.
• Microcentrifuge for 5 min and then very carefully remove 500 ul of the upper aqueous phase into a fresh eppendorf tube. Be
particularly careful NOT to remove any of the organic interphase. Remember..... don't get greedy, leave behind 100 ul of the
aqueous layer, there is plenty of DNA in the first 500 ul.
• To the aqueous layer add 500 ul of Chloroform:Isoamyl Alcohol (24:1) vortex vigorously, microcentrifuge for 2 minutes
and remove 450 ul of the top aqueous layer into a fresh eppendorf tube. (This step will remove any contaminating phenol).
• Precipitate out plasmid DNA from the 450 ul aqueous layer by adding 500 ul of isopropyl alcohol and allowing the sample
to sit at room temperature for 5 mins.
• Pellet plasmid DNA by microcentrifugation for 5 mins.
• Aspirate the isopropyl alcohol, recentrifuge briefly and reaspirate any trace amount of alcohol. Air dry the plasmid DNA
pellets at room temperature for 20-60 mins.
• Redissolve the plasmid DNA in 50 ul of double distilled water. Add 1 ul RNase A (10mg/ml) to remove residual RNA.
• Quantitate DNA and prepare sequencing samples.
MICB Sequencing Facility, July 2011 - page 1 of 7
ABI PRISM 310 SEQUENCING OF PLASMID DNA
DNA Template Preparation for Submission
TIPS:
1. If your plasmid preparation includes a phenol/chloroform extraction be careful to remove all traces of phenol and chloroform as
these will inhibit the sequencing reaction.
2. Amount of plasmid DNA to use depends on the size of the plasmid. The larger the plasmid being sequenced the smaller the
moles of plasmid present. Therefore use the following as a guideline:
plasmid size 2-3Kb
use 150 ng/ul
plasmid size 3-5Kb
use 200 ng/ul
plasmid size 5-8Kb
use 300 ng/ul
plasmid size > 8Kb
use 400 ng/ul
3. Pay attention to your pipetter. Monitor the pipetting to confirm that the amounts look correct.
4. Sequencing data will start 20-50+ nucleotides from the 3’ end of the primer site.
5. Avoid EDTA and use only pure ethanol for precipitation, no bulk ethanol.
6. After precipitating the sequence fragments, remove ALL the ethanol, DO NOT SIMPLY ALLOW IT TO EVAPORATE
OFF. You should pipette off the ethanol such that there should be NO ethanol visible anywhere in the tube. If there is ethanol
in the tube the sequencing will fail. If ethanol was allowed to evaporate from the tube then you will see an artifact thymidine (Tred) curve early in your sequence and a “dirty” start to your sequencing.
Primer Selection
•
•
Eurofins MWG Operon offer 50+ in-house sequencing primers FREE of CHARGE. See the complete list below.
If you need to prepare your own sequencing primer then YOU WILL NEED TO CONFIRM that your primer is suitable ie that it
does not form primer-dimer pairs, that it does not self compliment, that it’s Tm is not too low etc. This is best done using a primer analysis program. For more info see http://www.biocenter.helsinki.fi/bi/bare-1_html/oligos.htm and try their FASTPCR
demo program. In general these are the preferred characteristics of your primer:
1)18-30 nt in length
2)40-60% GC content
3)Tm between 50oC-70oC
4)No primer-dimer pair formation particularly at the 3’ end of the primer
5)Confirm that the primer is unique within your sequence
Remember that even though your primer meets all the above criteria THERE ARE NO GUARANTEES it will work. The ultimate test is the sequencing.
•
If your sequencing primer is NOT ideal then you can try to increase the primer concentration in the sequencing reaction from 5
pmol to 10 pmol.
MICB Sequencing Facility, July 2011 - page 2 of 7
MICB ABI PRISM 310 SEQUENCING GUIDE
SEQUENCING OF PCR PRODUCT DNA
PCR Product Analysis
Sequencing from PCR products is usually more problematic than sequencing from a plasmid. None-the-less by using a clean PCR
product and a good primer excellent sequence will be obtained. The most important aspect of PCR product sequencing is to ensure
that the PCR product is a pure, single band of sufficient quantity. This can be done by running part of the product on an 1-3% agarose gel, staining with ethidium bromide (EtBr) or SYBR Green to view the PCR DNA (Fig 1). This gel should be loaded carefully
as it will later be used to predict the relative amount of purified product that will be used for sequencing. You should avoid sequencing product where:
1) Multiple PCR product are observed (eg lanes labelled * in Fig 1)
2) Significant artifactual smears are observed (eg lanes labelled # in Fig 1)
3) Where the PCR product band is diffuse (eg lanes labelled @ in Fig 1)
4) Where the PCR product band is very faint (eg lanes labelled & in Fig 1)
5) One band – two products? (eg lanes labelled + in Fig 1)
Fig1. PCR Products (5 of 50ul) separated on an EtBr stained agarose gel
&&
&#
#
0.5
1
2
&* +
#
* * # #
& *@
* * @ @& &
0.5
2
1
0.5
Cleaning Up PCR Products
Gel Elution:
The “classic” method of purifying PCR products is to do GEL ELUTION. The band of interest is excised, PCR DNA separated from
the agarose, ethidum bromide removed and purified PCR DNA reprecipitated. Commercial kits like Qbiogene’s Clean-Gene II (Cat.
# 1001-400) are available for this purpose. Note that there is a size limit. Alternatively, the PCR DNA band can be eluted into a well
containing 5x TBE that has been cut out in front of the PCR DNA band. Ethidium bromide is then extracted from the PCR DNA
using isoamyl alcohol and purified PCR DNA is then reprecipitated. In both cases the procedure is relatively long and the amount of
PCR DNA must be relatively large; however, if a PCR fragment MUST be sequenced and multiple bands are present then this may
be the only way.
ExoSAP-IT Kit (US78200) for rapid efficient clean-up of PCR DNA from large numbers of tubes / wells:
from Amersham Pharmacia Biotech [Available from the MICB BioBar]
ExoSAP-IT Procedure:
To 5 ul of your PCR product add 1 ul ExonucleaseI and 1 ul Shrimp Alkaline Phosphatase. Incubate at 37oC for 15 min
and then 80oC for 15 min. You would then use 0.5-2 ul of this ExoSAP purified PCR product for sequencing. The amount is
dependent on the intensity of the PCR product band on the ethidium bromide stained gel (5ul of a 50ul rxn). (See circled
samples in Fig 1. Numbers below indicate microlitres (ul) ExoSAP purified PCR product to be used for sequencing).
MicroClean Kit (gel Company, 2MCL-05) for rapid cheap clean-up or PCR DNA from moderate number of tubes / wells:
MicroClean Procedure:
Add an equal amount of microclean reagent (MC) to PCR product (eg. 5ul MC to 5ul PCR product). Spin at 12-14000 rpm
for 7 minutes. Pipette all liquid off carefully. Add back 12ul ddH2O. Use 5ul for sequencing reaction.
MICB Sequencing Facility, July 2011 - page 3 of 7
ABI PRISM 310 SEQUENCING OF PCR PRODUCT DNA
PCR Template Preparation for Submission
TIPS:
1. Purify your PCR DNA (see Page 1 of 3 Cleaning up PCR products)
2. Amounts of purified PCR DNA to use depend on the size. The larger the PCR product being sequenced the smaller the moles
present. Therefore use the following as a guideline:
PCR product size 100-200bp
PCR product size 200-500bp
PCR product size 500-1000bp
PCR product size 1000-2000bp
PCR product size >2000bp
ExoSAP-IT cleaned PCR product
3.
4.
5.
6.
use 1-3 ng/ul
use 3-10 ng/ul
use 5-20 ng/ul
use 10-40 ng/ul
use 20-50 ng/ul
use 0.5-2ul (See Fig.1 & ExoSAP-IT on page 1of 3)
Pay attention to your pipetter. Monitor the pipetting to confirm that the amounts look correct.
Sequencing data will start 20-50+ nucleotides from the 3’ end of the primer site.
Avoid EDTA and use only pure ethanol for precipitation no bulk ethanol.
After precipitating the sequence fragments, remove ALL the ethanol, DO NOT SIMPLY ALLOW IT TO EVAPORATE
OFF. You should pipette off the ethanol such that there should be NO ethanol visible anywhere in the tube. If there is ethanol
in the tube the sequencing will fail. If ethanol was allowed to evaporate from the tube then you will see an artifact thymidine (Tred) curve early in your sequence and a “dirty” start to your sequencing.
REQUEST FORMREQUEST FORM.
Sequencing Primer Selection
Q– Can I sequence using the same primers used to generate the PCR product?
A– Ideally you would want to use a nested primer distinct from the PCR primer; however, in many cases using the same primer will
give good sequence. The primer (particularly it’s 3’ end) must be able to bind the PCR product DNA strongly to initial elongation. If
the end of the PCR product DNA obscured by tertiary folding and if the primer can not bind well to the template under sequencing
conditions, then no sequence will be obtained. REMEMBER that elongation parameters in sequencing could be significantly different from those of the PCR. Ultimately the quality of the DNA, the quality of the primer and the sequencing elongation parameters
will determine whether satifactory sequence will be obtained.
Primer Selection
•
If you need to prepare your own sequencing primer then YOU WILL NEED TO CONFIRM that your primer is suitable,- that it
does not form primer-dimer pairs, that it does not self compliment, that it’s Tm is not too low etc. This is best done using a primer analysis program. For more info see http://www.biocenter.helsinki.fi/bi/bare-1_html/oligos.htm and try their FASTPCR
demo program. In general these are the preferred characteristics of your primer:
•
1)
2)
3)
4)
5)
18-30 nt in length
40-60% GC content
Tm between 50oC-70oC
No primer-dimer pair formation particularly at the 3’ end of the primer
Confirm that the primer is unique within your sequence
Remember that even though your primer meets all the above criteria THERE ARE NO GUARANTEES it will work. The ultimate test is the sequencing.
•
If your sequencing primer is NOT ideal then you can try increase the primer concentration in the sequencing reaction from 5
pmol to 10 pmol.
MICB Sequencing Facility, July 2011 - page 4of 7
MICB ABI SEQUENCING GUIDE
BASICS ON ELECTROPHEROGRAMS
Overall Profiles
The three principle sequencing profiles are shown below.
Figure 1: Good Sequence Profile
Strong, but not excessive, peaks initially gradually decreasing in height.
Figure 2: Too Much DNA Sequence Profile
Excessive peaks initially with a rapid decline
after some 130 bp. The excessive initial peaks
will tend to obscure the start of the sequence.
T
–
Figure 3: Failed Sequence Profile
Failure may be due to any of the factors listed:
1. Too little DNA template
2. Poor or degraded primer
3. Poor reaction setup and execution
4. Poor loading of sample on the sequencer
5. Problem with the sequencer
*For EXTERNAL samples we control for 3-5
Artifact
With the BIGDYE reaction mix there is a propensity to have a large T (red-peak) artifact in the 200-230 nt region. This artifact can
be removed with repeated ethanol precipitations. The presence and intensity of the T-artifact peak is a good indicator of how well or
poorly ethanol is being removed during ethanol purification of elongated sequencing fragments.
Figure 4: No T– artifact
Figure 5: Small T– artifact
Figure 6: Large T– artifact
Assessing Ambiguous (N) Nucleotide
If tertiary structure causes specific sequence fragments to run faster or slower than predicted, the ABI software will generate an N.
This problem can easily
be seen on the electropherogram and the
correct nucleotides can be
manually assigned.
Figure 7: TNNAG is really TCAAG
Figure 6: T– artifact obscured sequence
ATTCTAATTC
MICB Sequencing Facility, July 2011 - page 5 of 7
NEW SEQUENCING REQUEST FORM MICB RM ON6042
Name: _________________ Phone #:______________ Account to Charge _____________________
Investigator:_________________ Dept.________________ Bldg.___________Rm#________
Date Submitted (mm/dd/yy): ___ / ___ / ___ E-mail ______________________
Sample Submission Guidelines (Tubes or Plates)
Tubes- Use 1.5mL (large) Eppendorf tubes. Label cap of each tube with sample name.
Plates– Use 96-well, v-bottom, full skirt PCR plates. Minimum 48 samples. G12 + H12 must be empty. Capped.
Express Plates– Plates as above. Template & CUSTOM primer or 10ul ddH2O (for empty wells). G12& H12 must be
empty. Completed Eurofin Plate Order or ExpressPlate Order Form must be e-mailed to [email protected]
Each tube or well will contain either 10 µL of template only OR 8 µL template + 4 µL CUSTOM primer.
*If you are short on template, remember that 5 µL is the minimal volume needed for ONE sequencing reaction.
Template Provided
Template
Concentration
(↑ with ↑template size)
Custom Primer
Concentration
Volume
Template Only
Volume
Template + Primer
(*5 µL min. vol.)
(*6 µL min. vol. 4 + 2 µL)
150-400 ng / µL
2 µM (pmol / µL)
10 µL (@150-300ng/µL)
8 µL + 4 µL (@2µM)
PCR (<300bp-Purified)
10-20 ng / µL
2 µM (pmol / µL)
10 µL (@10-20ng/µL)
8 µL + 4 µL (@2µM)
PCR (300-1000bp-Purified)
20-40 ng / µL
2 µM (pmol / µL)
10 µL (@20-40ng/µL)
8 µL + 4 µL (@2µM)
PCR (>1000bp-Purified)
30-60 ng / µL
2 µM (pmol / uL)
10 µL (@30-60ng/µL)
8 µL + 4 µL (@2µM)
Plasmid Normal(<20kbp)
Tube #
Eg.
1
Sample Name
(Max 8 characters)
Eg. DD1-CST1
Primer Code (50+ FREE see page 2)
or CUSTOM (your primer)
Eg. T3 or T7 or SP6 or CUSTOM
Template Type
PCR or Plasmid
Barcode
(office use only)
Check one
1
PCR
Plasmid
2
PCR
Plasmid
3
PCR
Plasmid
4
PCR
Plasmid
5
PCR
Plasmid
6
PCR
Plasmid
7
PCR
Plasmid
8
PCR
Plasmid
9
PCR
Plasmid
10
PCR
Plasmid
11
PCR
Plasmid
12
PCR
Plasmid
13
PCR
Plasmid
14
PCR
Plasmid
15
PCR
Plasmid
16
PCR
Plasmid
Bring samples and completed form(s) to ON6042 by noon. Put samples in freezer RED RACK.
Initial shipments on MONDAY and WEDNESDAY of every week. SHIPPING is FREE.
Results will be placed on the SEQUENCING SERVER in 2-3 days or e-mailed directly.
MICB Sequencing Facility, July 2011
NEW SEQUENCING +50 FREE PRIMERS MICB RM ON6042
Primers available in-house at no additional cost. Enter the primer code on Request Form
#
Primer
Code
Primer sequence
5’ - 3’
#
Primer
Code
Primer sequence
5’ - 3’
1
3AOX
GCA AAT GGC ATT CTG ACA TCC
29
petup
ATG CGT CCG GCG TAG A
2
5AOX
GAC TGG TTC CAA TTG ACA AGC
30
pFBACf
TCC GGA TTA TTC ATA CCG TCC C
3
96glll
CCC TCA TAG TTA GCG TAA CG
31
pFBACr
CCT CTA CAA ATG TGG TAT GGC TG
4
CMVf
CGC AAA TGG GCG GTA GGC GTG
32
pGexF
ATA GCA TGG CCT TTG CAG G
5
GadFor
GGG ATG TTT AAT ACC ACT AC
33
pGexR
GAG CTG CAT GTG TCA GAG G
6
GadRev
AAG AAA TTG AGA TGG TGC AC
34
pGLfor
GTA TCT TAT GGT ACT GTA ACT G
7
Gal4AD
TAC CAC TAC AAT GGA TG
35
pGLrev
CTT TAT GTT TTT GGC GTC TTC C
8
Gal4BD
TCA TCG GAA GAG AGT AG
36
pGL3for
CTA GCA AAA TAG GCT GTC CC
9
M13R
CAG GAA ACA GCT ATG ACC
37
pJET12F
CGA CTC ACT ATA GGG AGA GCG GC
10
M13R49
GAG CGG ATA ACA ATT TCA CAC AGG
38
pJET12R
AAG AAC ATC GAT TTT CCA TGG CAG
11
M13F
TGT AAA ACG ACG GCC AGT
39
pJET1F
GCC TGA ACA CCA TAT CCA TCC
12
M13F43
AGG GTT TTC CCA GTC ACG ACG TT
40
pJET1R
GCA GCT GAG AAT ATT GTA GGA GAT C
13
malE
GGTCGTCAGACTGTCGATGAAGCC
41
pQEfPR
GTA TCA CGA GGC CCT TTC GTC T
14
pBabeF
TGA CCT GGG AAG CCT TGG CT
42
pQErev
CAT TAC TGG ATC TAT CAA CAG GAG
15
pBabeR
TTG CTG ACT AAT TGA GAT GCA TGC TTT
43
pShCMVf
GGT CTA TAT AAG CAG AGC TG
16
pBadF
ATG CCA TAG CAT TTT TAT CC
44
pShCMVr
GTG GTA TGG CTG ATT ATG ATC AG
17
pBadR
GAT TTA ATC TGT ATC AGG
45
TrcHisF
AAT CTG TGT GGG CAC TCG
18
pcDNAF
GGC TAA CTA GAG AAC CCA CTG
46
TrcHisR
CTT CTG CGT TCT GAT TTA ATC TG
19
pcDNAR
GGC AAC TAG AAG GCA CAG TC
47
RV3
CTA GCA AAA TAG GCT GTC CCC
20
BGHrev
TAG AAG GCA CAG TCG AGG
48
SP6
CA TTT AGG TGA CAC TAT AG
21
EGFP35r
AGG TTA AGT AAA GCG TCT G
49
T3
AAT TAA CCC TCA CTA AAG GG
22
EGFP35u
ACC GTA AGT AGC ATC ACC TTC
50
T7
TAA TAC GAC TCA CTA TAG GG
23
EGFP36r
TGG TCT TGT TAG AAT TTG TTA C
51
T7term
CTA GTT ATT GCT CAG CGG T
24
EGFP36u
CTT TTC GGT TAG AGC GGA TGT G
52
V5
ACC GAG GAG AGG GTT AGG GAT
25
EGFPC1F
GAT CAC TCT CGG CAT GGA C
CUSTOM
Your personal primer added to Template
26
EGFPC1R
CAT TTT ATG TTT CAG GTT CAG GG
27
EGFPN1F
GTC GTA ACA ACT CCG CCC
28
EGFPN1R
GTC CAG CTC GAC CAG GAT G
MICB Sequencing Facility, July 2011