Sequencing Instructions and On-line Sequencing Form

Plasmids

• Provide DNA as described above. We can supply some universal primers such as T3, T7, SP6, M13 Forward and Reverse if you desire or you can add them yourself . Please note that the maximum volume of your DNA sample has to be 8.4 ul, 10ul if the sequencing primer is added.
• Complete and submit the electronic sequencing form if you are on campus, then bring your samples to room 807, A Tower Building, Health Sciences Campus (500 South Preston St.). Give to Xiaohong Li or place in the drop-off cooler outside the lab door. If you are off-campus, please contact Xiaohong Li about the procedure for handling samples.
• Sequences will usually be completed within 48-72 hours (we try to get them out sooner, but that depends on how much volume we are sequencing at the time). Sequence files will be forwarded to you by e-mail. In general, we will send both "seq" (text file) and "scf" files. The "scf" file contains the image data so you can look at the quality of the sequence. All sequence handling programs can read these files. Programs like Vector NT, DNAStar and GCG are now available, please contact us if you are interested in access to these programs.
  
• If you need additional details of methods used by us for sample preparation and sequencing, please see the Beckman Coulter protocol posted here with their permission.

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BCI P/N 608118-AA
CEQ 2000
Dye Terminator Cycle Sequencing with Quick Start Kit

Storage of the CEO Cycle Sequencing kit must be in a -20° C non-frost free freezer.
1. Preparation of the DNA sequencing reaction*:

Prepare sequencing reaction in a 0.2 mL thin-wall tube or microplate well. All reagents should be kept on ice while preparing the sequencing reactions and should be added in the order listed below.

dH20 (to adjust total volume to 20µL)	0 - 9.5 µL
DNA Template** (See Template Preparation)	0.5 - 10.0 µL
Customer supplied or -4 / Sequencing Primer (1.6 pmol/µL or 1.6µM)	20.0 µL
DTCS Quick Start Master Mix	8.0 µL
TOTAL	20.0 µL

**Use 0.5 µL for pUC18 control template.
*Note: Mix reaction components thoroughly. Consolidate the liquid in the bottom of the tube or well by briefly centrifuging before thermal cycling.

2. Thermal cycling program for 30 cycles followed by holding at 4°C :

96°C	20 sec.
50°C	20 sec.
60°C	4 min.

3. Ethanol precipitation: Precipitation in Individual Tubes

1. Prepare a labeled, sterile 0.5 mL microfuge tube for each sample.
2. To each labeled tube add 4µL Stop Solution (1.5 M NaOAc + 50 mM EDTA prepared fresh daily by mixing equal volumes of the 3M NaOAc and 100mM EDTA listed in "Required materials not provided by Beckman Coulter") and 1 µL of 20 mg/mL glycogen (supplied with the kit).
3. Transfer the sequencing reaction to the appropriately labeled 0.5 mL microfuge tube and mix thoroughly.
4. Add 60 µL cold 95% (v/v) ethanol/dH2O from -20°C freezer and mix thoroughly. Immediately centrifuge at 14,000 rpm at 4°C for 15 minutes. Carefully remove the supernatant with a micropipette (the pellet should be visible). Note: For multiple
   samples, always add the cold ethanol/dH20 immediately before centrifugation.
5. Rinse the pellet 2 times with 200 µL 70% (v/v) ethanol / dH20 from -20°C freezer. For each rinse, centrifuge immediately at 14,000 rpm at 4°C for a minimum of 2 minutes. After centrifugation carefully remove all of the supernatant with a micropipette.
6. Vacuum dry for 40 minutes.
7. Resuspend the sample in 40 µL of the Sample Loading Solution (provided in the kit).
   

   Precipitation in the CEO 2000 Samples Plates
   The Thermal Cycling and Ethanol precipitation can be performed in the CEO 2000 sample plate. For instructions, see pages 19-20 in the "CEO™2000 Dye Terminator Cycle Sequencing Chemistry Protocol" (P/N 718119AB).

4. Sample preparation for loading into the CEO:
a) Transfer the resuspended samples to the appropriate wells of the CEO sample plate (PIN 609801).
b) Overlay each of the resuspended samples with one drop of light mineral oil (provided in the kit or Sigma Cat # M 5904).
c) Load the sample plate into the CEO and start the desired method.

Template Preparation

1. DNA Template preparation:
Prepare sufficient template to allow for its accurate quantitation and purity verification. The quality of the DNA template will depend upon the procedure and the source of the DNA used. The following are the recommended protocols:

• QIAGEN QIAwell™ and QIAprep™ DNA isolation protocols (dsDNA and ssDNA)
• QIAGEN QIAquick™ PCR purification protocol (PCR products).
  Note: Determine the quality and quantity of template DNA by agarose gel electrophoresis.

2. DNA Template amount:
The amount of template DNA to use in the sequencing reaction depends on the form of the DNA (dsDNA plasmid, ssDNA M13, PCR product, etc.). It is important to accurately quantitate the amount (moles) of DNA when performing the DNA sequencing reaction (see formula and table below for details). The molar ratio of primer to template must be greater than or equal to 40:1. Listed below are the recommended amounts of DNA:

dsDNA	0-100 fmol
ssDNA	25-50 fmol
Purified PCR products	25-100 fmol

The following table can be used to estimate DNA concentrations.
Table for estimating the dsDNA concentration.

Size (kilobasepairs)	ng for 25 fmol	ng for 50 fmol	ng for 100 fmol
0.2	3.3	6.5	13
0.3	4.9	9.8	20
0.4	6.5	13	26
0.5	8.1	16	33
1.0	16	33	65
2.0	33	65	130
3.0	50	100	195
4.0	65	130	260
5.0	80	165	325
6.0	100	195	390
8.0	130	260	520
10.0	165	325	650
12.0	195	390	780
14.0	230	455	910
16.0	260	520	1040
18.0	295	585	1170
20.0	325	650	1300
48.5	790	1500*	1500*

For ssDNA, the values (ng) should be divided by 2.
*Use no more than 1.5 µg of template DNA.

3. Template Pre-Heat Treatment
For certain plasmid DNA templates, the following pre-heat treatment improves both signal strength an current stability.

• Dilute the template with water to the appropriate concentration.
• Heat the template at 96°C for 1 minute in a thermal cycler and then cool to room temperature before adding the remainder of the sequencing-reaction components.
• Do not add any other sequencing-reaction components to the plasmid template before performing this pre-heat treatment.
• If the raw data signal declines steeply when using this treatment, change the heating conditions to 86°C for 5 min. If the current is low or unstable following this treatment, increase the treatment to 96°C for 3 min.

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Appendix
Appendix A
Sequencing of PCR products

• All PCR products must be homogeneous in size as judged by gel electrophoresis.
• Purified PCR products
  a) Remove unincorporated primers and dNTPs using QIAGEN QIAquick™ PCR purification system.
  b) Use 25-100 fmol of PCR product and 3.2 pmoles of primer.

Unpurified PCR products

a) For the original PCR amplification, the primer concentration should be 0.2 µM or less, while the dNTP concentration should be 50 µM or less.
b) The amplification should be sufficient to produce a concentration of amplified fragment that is greater than or equal to 10 fmol/µL
c) Dilute this amplified fragment approximately 10 fold to result in a concentration of greater than or equal to 1 fmol/µL.
d) Use 5-15 fmol of this diluted, unpurified PCR product and 3.2 pmoles of primer.

Appendix B

Sequencing of Large Templates
Adding 50-100 fmol for large templates such as BACs, cosmids and
PACs is impractical. The following procedure should be used when sequencing large templates.
1. Use 1.5 µg of the template in 6µL of sterile, deionized water.
2. Pre-heat the template at 96°C for 1 minute.
3. Add the sequencing-reaction components as described in the standard protocol.
4. Cycle for 50 cycles using the appropriate cycling conditions for the primer being used.
5. Precipitate with ethanol, as previously described.

Appendix C

• Store the Sample Loading Solution in 350 µL aliquots at -20°C in a non frost free freezer.
  
• Use each aliquot only once - do not freeze/thaw the Sample Loading Solution.

This information is from the CEQ Quick Start Kit, document number 608118-AA and is posted with permission from Beckman Coulter Inc.