Laragen Protocol (adapted from www.laragen.com for Simpson lab; Don, 3’02; revised 4-04)
Cycle sequencing reactions
To prepare the reaction mixtures, use following amount of DNA depending on
templates (Adapted from PE Biosystems):
Step
Action
For each reaction, add the following reagents to a separate tube:
Reagents
Quantity
single strand DNA
50 - 100 ng
200 - 500
Template
double strand DNA
ng*
1
PCR product DNA
30 - 90 ng
Primer
5 pmol**
BigDye Mix (ABI sequencing Mix)
4 ul
Deionized water
q.s
Total Volume
10 ul
2
Mix well and spin briefly
*2 ul of a Qiagen miniprep (eluted with 50 ul)
** 1 ul of 5 pmoles/ul
Typically, 2 ul miniprep DNA; 0.5 ul primer; 4 ul mix; 3.5 ul water.
Blue mix is in “front” room freezer in a round “portable freezer” marked ABI. Finish
one tube before starting another. Order more when down to last tube.
Stocks of standard primers are in a cardboard box in the ”restriction enzyme” freezer
in back room. Note also that Laragen will provide primers free of charge (list
below)—just leave a note with your samples going out or send an e-mail. You can
use also almost any primer for sequencing. Follow the rules for a PCR primer with a
Tm of about 55°C or adjust the annealing temperature (50°C in the protocol below).
Cycle sequencing parameters for GeneAmp PCR System
The program is already set up as protocol 149 on the PE unit closest to the aisle. To set up
cycle sequencing reaction with the GeneAmp PCR System (2400, 9600, 9700) for plasmids
or PCR products, use the follow parameters (Adapted from PE Biosystems):
Action
Step
1
Place the tubes in a thermal cycler and set the
volume to 10 ul.
1 min 96°C
Repeat the following for 25 cycles:
* Rapid thermal ramp to 96°C
* 96°C for 10 sec.
2
* Rapid thermal ramp to 50°C
* 50°C for 5 sec.
* Rapid thermal ramp to 60°C
* 60°C for 4 min.
Rapid thermal ramp to 4°C and hold until ready
3
to purify.
Spin down the contents of the tubes in a
4
microcentrifuge.
5
Proceed the purification steps.
Note: Rapid thermal ramp is 1°C per sec.
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The run takes approximately 2.5 hours. Finished samples can be stored overnight
at refrigerator temperatures or below. They can be stored at least several days
frozen.
Purify cycle sequencing products
There are two techniques you can use to purify the cycle sequencing products. i.e.
Isopropanol Precipitation and Spin Column .
Isopropanol Precipitation
Adapted from PE Applied Biosystems Protocol
1. For 10 ul of cycle reaction, add 10 ul of sterile distilled water to make
the final 20 ul volume.
2. Add 80 ul of 75% isopropanol and mix gently. The final isopropanol
concentration should be 60%. Note: This procedure does not use salt.
3. Leave the tubes at room temperature for 20 minutes to precipitate the
extension products. Note: Precipitation times < 15 minutes will result in
the loss of very short extension products. Precipitation > 24 hours will
increase the precipitation of unincorporated dye terminators.
4. Centrifuge at top speed (ca 12,000 rpm) with a desktop centrifuge for
15 minutes. Aspirate as much isopropanol in the tubes as possible.
5. Add 250 ul of 75% of isopropanol and centrifuge at top speed (ca
12,000 rpm) with a desktop centrifuge for 5 minutes. Aspirate as much
isopropanol in the tubes as possible.
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6. Air dry or SpeedVac dry the tubes. The tubes are now ready for
sequencing run.
Spin Column (stored on top-right shelf of sliding-door refrig.)
We recommend AGTC Gel Filtration Cartridges from Edge BioSystems (Cat
42453).
7. Centrifuge the AGTC gel filtration cartridges as supplied for 2 minutes
at 750x g (3000 rpm in microfuge. Not full speed.)
8. Transfer the cartridge to a clean the microcentrifuge tube provided with
the kit.
9. To 10 ul of cycle reaction, add 10 ul of sterile distilled water to make
the final 20 ul volume. and add samples to the packed column. Be sure
the fluid runs into the gel.
10. Loosen the cap and centrifuge for 2 minutes at 750x g. Retain eluate.
11. Speedvac dry the tubes and which are now ready for sequencing run.
[Medium heat; 15 min. Remove promptly.]
Ordering sequence reaction pickup:
Go to Laragen site/ www.laragen.com.
Enter Client ID:
Investigator is Larry Simpson
Enter your (first) name, and place your samples in the “Laragen” freezer labeled with
your name.
Sequences are $5 each. Enter order in both the online sequencing spreadsheet,
and the total cost in the order book. Orders placed before 10 AM will be picked up in
the Simpson lab between 1 and 3 the same day.
What about my sequencing primers?
primers, you can order them from any major oligo manufacturers. Generally
speaking, the oligonucleotides used as sequencing primers do not need extra
purification and desalting. The working concentration for sequencing primers should
be at 10 pmole/ul.
M13 Forward
M13 Reverse
T7 Promoter
T3
SP6
T7 Terminator
Standard Primer Sequences
GTA AAA CGA CGG CCA GT
CAG GAA ACA GCT ATG AC
TAA TAC GAC TCA CTA TAG GG
ATT AAC CCT CAC TAA AG
ATT TAG GTG ACA CTA TAG
CTA GTT ATT GCT CAG CGG TG
3
pGEX 5'
pGEX 3'
RV3
BGH Reverse
GGG CTG GCA AGC CAC GTT TGG TG
CCG GGA GCT GCA TGT GTC AGA GG
CTA GCA AAA TAG GCT GTC CCC
TAG AAG GCA CAG TCG AGG C
How can I retrieve my sequences?
ftp://sim102@ftp.laragen.com
PW: 4dna*
address and FTP site. We do not recommend sending large number of sequences
via email.
How many base pairs can I get from a single run?
- 650 bp in a single run.
Based on our experience, most of our sequences are above 575 bp.
How close can I read from the primer?
17 - 25 bp away from the the primer. With good, clean template DNA and
reasonably fresh primers, 600 nt of flawless sequence is possible.
How can I ship my sample to Laragen?
mailling address is Laragen, Inc., 10755 Venice Blvd, Los Angeles, CA 90034.
What is the turnaround time?
Type of Sequencing
Turnaround Time
Ready-to-run
24 hours
Sequencing from purified templates
48 hours
Full services sequencing
72 hours
How long do you keep my DNA samples and primers?
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Unless you specifically request, we normally keep our clients' DNA samples and
primers for 3 weeks. The DNA samples and primers will be trashed after that.
From Laragen site: www.laragen.com
Why didn't my sequencing work? DNA sequencing reactions may fail due for
different reasons.
Poor quality of template DNA: Contaminants in the DNA prep will interfere with
the reaction and cause failures, noise and miscalls. Contaminants are ethanol,
protein, salt, PEG and genomic DNA.
Not enough DNA: Insufficient DNA will lead to insufficient signals or failed
reactions. On the flip side, too much DNA will also interfere with the reaction.
Primer concentration: 5 pmole of primer is required per sequencing reaction.
Make sure your primer concentrations are correct. Too much or too little primer may
result in sequencing failures. Based on our experience, you should test your
sequencing primer as a PCR primer first. If the primer works as a PCR primer, it
usually works fine as a sequencing primer.
Difficult Templates: Some templates are difficult to sequence. For example, it is
difficult to sequence a GC righ DNA template with regular BigDye premix. If that is
the case, we will use dGTP BigDye chemistry to get around it.
What is your policy for repeat sequences?
Mistakes do happen. If the DNA sequencing reactions fail because of our error,
we will repeat the reaction for free. However, if the sequencing reactions fail due to
the quality of templates and/or primers, we will charge the sequencing reaction if the
customer requests for repeat.
[Software for reading sequences is available on the Laragen Website,
www.laragen.com.]
Laragen, Inc. is located at 10755 Venice Blvd., West Los Angeles, CA 90034. We
are at the northwest corner of Venice Blvd. and Selby St. and only about 15 minutes
from UCLA campus.
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