The DNA Sequencing/Solexa Core utilizes “dnaTools,” a Web-based lab management software. This software allows investigators to submit DNA sequencing service requests
from their lab computers directly to the DNA Sequencing Lab. After each data collection run, the data is automatically processed and uploaded to a server for immediate retrieval by the investigator. The DNA analyzer is equipped with an automatic plate loader and sample injection system, which provides continuous sample throughput for up to 16 96-well sample plates. The combination of dnaTools and DNA analyzer automation provides 24-hour data collection, analysis and retrieval.
Template - PCR Product
Concentration at least 0.5 ng per 100 base pairs for fragments less than 500base pairs
Dilute PCR sample to 1 ng/ulif your PCR fragment is less than 500 base pairs
If your PCR sample is greater than 500base pairs then dilute PCR to 3 ng/ 100 base pairs in 2 ul volume.
For example: If your PCR size is 600 base pairs, multiply 600 X 0.03 which will give you the total amount of DNA necessary (18 ng) for sequencing.Then dilute your PCR sample to the correct working concentration of 9 ng/ul in 2 ul total volume.
Template – BAC Clones
1 ug/ul concentration for BAC DNA
Template – Plasmid
Concentration at 100 ng/ul
2.0 ul needed per reaction
Concentration at 1-2 pmol/ul
10 pmol/ul for BAC sequencing primers
4 ul needed per reaction
Entry Form (After you have clicked Enter DNA Sequencing Requests, and entered the number of reactions)
City of Hope
users, leave the PO Number field blank.This is for outside users only.
Under the Grant Code Num. field please enter 1111.
The account number you enter must use the subaccount of 8028. Do not change this. Enter your entire account number (20/30 – 8028 – 6 digit account number) Ex. 30-8028-xxxxxx
Enter the PI name “last name, first name” and in all capitals. Ex: SMITH, JOHN
BigDye_V3.1 – For basic sequencing.Can read out to approximately 900 base pairs.
BigDye_V1.1 – Used only for short (less than 500 base pairs) PCR products when high resolution near the priming site is needed.
BigDye_3.0-dGTP – Used only for templates that are likely to form strong secondary structures, hairpin loops, or with high GC content (greater than 75%) or bisulfite-treated samples. When using this chemistry, please include the Tm of your primer in the comments box.
Make sure after submitting the entry form, you print 2 copies of the table that appears. One of these copies must be placed in the inbox to inform us that the samples are ready to go. If we do not receive a copy of this form, your samples will never be sequenced. The other copy is for your reference.
Please label your samples with less than 8 characters if possible.Write the labels on the cap of the tubes you are submitting.Make sure the labels on your tubes are identical to the labels on the order form.