Q1: How much DNA do I need? | ||
A1: Samples should be submitted in a total volume of 12 µl of DNA and primer mix. Sequencing Handout Plasmid plus insert < 5kb between 20-45 ng/ul, > 5kb between 50-100 ng/ul; A good rule to follow is 3 ng/ul per kb low end, 10 ng/ul per kb on the high end. PCR 2 to 5 ng/100 bp* (500bp needs 10 to 50 ng total) ssDNA 50 - 100 ngBACs and Cosmids 1 - 2 µgBacterial Genomic DNA 2 - 3 µg | ||
Q2: How should I quantify my DNA? | ||
A2: Using a spectrophotometer like the NanoDrop is the quick and easy way to quantitate samples. But it is always best to run the samples on an agarose gel to verify concentration. Methods for Examining DNA Quality and Quantity:
| ||
Q3: How much primer do I need? | ||
A3: 5 to 20 pmol. We use 10 pmol. | ||
Q4: Primers provided by the Sequencing Facility? | ||
A4: Yes we have the following primers we offer with no charge: M13 Forward (-20) 5'-GTA AAA CGA CGG CCA GT-3' | ||
Q5: In what type of tube should I send the sample? | ||
A5: Samples should be submitted in 1.5ml microfuge tube. | ||
Q6: How do I submit a sequencing request? | ||
A6: Sequencing request should be submitted on our dnaLIMS website, at http://coreweb.icmb.utexas.edu ( http://146.6.133.14).For more information see the dnaLIMS Instructions. | ||
Q7: Where do I bring my samples? | ||
A7: Samples should be brought to MBB 1.426. There is a cold box on the west wall. Samples should be placed in the green racks. Facility Map | ||
Q8: Where is the Sequencing Facility? | ||
A8: We are located in Room 1.426 in the MBB (Moffett Molecular Biology Building) See the Facility Map or the Campus Map: | ||
Q9: When do the samples need to be in the Sequencing Facility to be run that day? | ||
A9: All samples that are in the Core Facility at 10 am will be run that day. | ||
Q10: When will I get my data? | ||
A10: The data is transfer is automated and should be available before 10 am the next day. | ||
Q11: How do I retrieve my data? | ||
A11: Sequencing data can be downloaded from our dnaLIMS website, at http://146.6.133.14.For more information see the dnaLIMS Instructions. | ||
Q12: How do I view my data? | ||
A12: The data can be viewed in the web browser. | ||
Q13: My samples failed, Why? | ||
A13: The #1 problem with samples that fail is template concentration. If you're having trouble with the spec in your lab, come down and use our NanoDrop. It's free and only takes a minute of your time and a microleter of your sample! Having the correct template concentration initially can save you a lot of time and money later.Contamination is another reason why samples fail. Contamination can be in the form of the following and in either your template, primer or both:Two templates present in one sampleRNAProteins To fix this problem you should do one or all of the following: | ||
Q14: I want you to add a Core provided primer for me. How do I let you know? | ||
A14: When you fill out your sequencing order online just be sure to check on radio button under "Service Requested" and "Core_prep that says "Template_Only." Also, make sure you click on one of the primers listed in the prime drop down menu. These are the primers we have available. The only way we know to add primer is if "Template_Only" is checked. Submit template only samples as you normally would. Just submit 11ul DNA at 40ng/ul. | ||
Q15: How long of sequence should I get? | ||
A15: We routinely get greater than 1000 bases. With quality samples over 900 bases is common. | ||
Q16: What are Phred Scores (Q20)? | ||
A16: Phred Quality Scores | ||
Phred quality score | Probability that the base is called wrong | Accuracy of the base call |
10 | 1 in 10 | 90% |
20 | 1 in 100 | 99% |
30 | 1 in 1,000 | 99.9% |
40 | 1 in 10,000 | 99.99% |
50 | 1 in 100,000 | 99.999% |
It has been shown that Phred's error probabilities are very accurate - if Phred assigns a quality score of 40 to a base, the chances that this base is called incorrectly are indeed just 1 in 10,000. This high accuracy has been observed for sequences generated at different laboratories, each using a different combination of sequencing enzymes, fluorescent dyes, and gel run conditions.
The high accuracy of Phred quality scores make them an ideal tool to assess the quality of sequences. The most commonly used method is to count the bases with a quality score of 20 and above (sometimes called "high quality bases") or 30 and above ("very high quality bases"). By looking at individual sequences, failed reactions or low-quality reads can easily be identified. When looking at collections of sequences, the effect of different sequencing methods on sequence quality can be directly measured. This allows straightforward quality control in sequencing projects, and can give easily available measures to optimize sequencing operations.
|
Q17: I am not getting good results what should I do? |
A17: Make sure you are using enough DNA, we request 150 to 300 nanograms for plasmids of 5kb size. Wrong DNA conentration (high or low) is the most common reason for sample failure. |
Q18: Will my samples be rerun if the results are not good? |
A18: We rerun samples that we believe that we might be able to improve the sequencing data. Samples can also be rerun by request. |
Q19: Who should I contact if I am having trouble with sequencing? |
A19: Email DNA Facility or come by MBB 1.426L or call 475-7844. After 1 pm is best time. |
Q20: How should I purify my DNA? |
A20: Most of the DNA from Kits work fine. Make sure the sample is resuspended in water, not TE. EDTA can cause lower signal intensities. Also for Qiagen do the optional wash to help get rid of any contaminates. Make sure the sample is dry before elution to prevent the presence of ethanol. |
Q21: What are the best primer conditions? |
A21: Primers should be at least 18 bases long to ensure good hybridization.Avoid runs of an identical nucleotide, especially runs of four or more Gs.Keep the G-C content in the range 30 - 80%, preferably 50 - 55%.Tm between 45 and 60°C is best. Primers with Tm>45°C produce better results than primers with lower Tm.Primers with a G-C content less than 50%, it may be necessary to extend the primer sequence beyond 18 bases to keep the Tm>45oC.Use of primers longer then 18 bases also minimizes the chance of having a secondary hybridization site on the target DNA.Avoid primers that can hybridize to form dimmers or palindromes.The primer should be as pure as possible, preferably purified by HPLC and resuspended in dH2O. |
Q22: What if I want to submit two samples with my own primer and two samples that I want the Core Facility to add primers to? |
A22: You need to submit two separate requests. On the first request enter the samples as "primer-template." On the second request enter the samples as "template." |
