Laragen Protocol (Adapted from for Simpson Lab; Don, 3- 02; Revised 4-04)

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 Template double strand DNA 200 - 500 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 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 to 3 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.

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. 6. Air dry or SpeedVac dry the tubes. The tubes are now ready for sequencing run.

2 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?

 We provide 5 universal sequencing primers free of charge. For customized 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.

Standard Primer Sequences M13 Forward GTA AAA CGA CGG CCA GT M13 Reverse CAG GAA ACA GCT ATG AC T7 Promoter TAA TAC GAC TCA CTA TAG GG T3 ATT AAC CCT CAC TAA AG SP6 ATT TAG GTG ACA CTA TAG T7 Terminator CTA GTT ATT GCT CAG CGG TG pGEX 5' GGG CTG GCA AGC CAC GTT TGG TG pGEX 3' CCG GGA GCT GCA TGT GTC AGA GG RV3 CTA GCA AAA TAG GCT GTC CCC

3 BGH Reverse TAG AAG GCA CAG TCG AGG C

How can I retrieve my sequences?

 Download your sequences from our server.

ftp://[email protected]

PW: 4dna*

 We can use email or FTP to send your sequences if you give us your email 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?

 Depends on quality of DNA templates, we can get 550 - 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?

 Depends on quality of DNA templates, we can get reliable sequence starting from 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?

 You can ship your DNA sample either in solution or dry pellet by mail. Our 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?

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.

4 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.