Detailed Sample submission instructions
1. Sanger Sequencing Submission Form:-
- It is very much important to fill all the details correctly and it is user’s responsibility to verify the sample and primer name as well as concentration before making submission to facility. If sample/primer name found different that are mentioned on form, facility will NOT contact user. The samples will be processed according to details on form/submission.
- Please count the number of reactions to be processed and fill correctly on respective box. One sample – one primer is considered as one reaction. For e.g. If you want to sequence your one sample with Forward and Reverse primer then total reaction number is two.
- If reaction number is more than 16 it is mandatory to upload excel file having detailed structure of reaction. The user needs to provide complete sample and primer details like type of sample (Plasmid/PCR), size of insert/product, concentration, volume etc.
- It is responsibility of user to provide correct framework of reaction(s). Please don’t provide all reactions in just one line. Please mention for each reaction the sample and primer(s) that needs to be sequence in orderly manner.
- Users are requested to keep sample or primer name short if possible. Please avoid usage of space and special characters, instead use hyphen or underscore for labeling of sample because Sanger Sequencing Software does not accept special character except these two.
- If sample is submitted in 8 well strips or 96 well plate please mentioned the same on respective field.
- If you have any additional notes like Quality of sample, Probable contaminant(s), Secondary Structure, GC/GT/GGG stretch, Repeats in your region of interest then it should be indicated in BOLD in the ‘Additional Notes’ or comment section. This will help us to counteract initially, resulting in saving time of both the parties and try to prevent failure in sequencing.
- Please provide the name of method used for plasmid or PCR product purification.
- Please check and confirm the details before submitting the form. Also ensure after clicking the ‘Submit’ button the form is uploaded or not. After submission the SS or Project ID is generated which has to be taken down by the user. Future discussion of results will be based on this project ID.
- Kindly submit the samples in facility as soon as the form is submitted. Failure in following this instruction leads to disturbance in order of the sequencing setup.
- Priority would be given according to order in which sample submission form is received. To use the instrument in the most effective manner and to minimize the instrument running cost, facility will attempt to collect full 96 samples before initiating a run.
- Please! Please! Note! It is MANDATORY to run 1.5ul of plasmid (concentration150ng/µL) and/or 2-3uL of PCR products (10-30ng/µL) on gel before submission to the facility. Users have to attach the same gel picture with the form. Because spectrophotometer is not reliable in terms of concentration!!!! Running on gel confirms the sample concentration. If not followed then facility will not be responsible if the sequencing failed due to concentration was not appropriate.
2. Sample Submission: -
- The sample(s) and primer(s) need to be submitted in zip lock bag only. The zip lock bags are kept near the door of facility.
- Please drop your sample in drop box kept in 4oC refrigerator near the facility entrance.
User has to strictly follow these instructions:
- The Zip lock bag should have the user’s name, Request No./Lab name/number.
- Most importantly the SS or Project ID should be written in BOLD on zip lock bag. The project ID is very important to identify user and their samples. It also prevents confusion of different users with same name and helps in easy grouping of samples according to one or many SS ID of same or different user(s).
- If user has not followed these instructions then samples won’t be processed even after submission of form.
- If PCR product is to be submitted for sequencing, then users has to purify the samples using Bead based purification method. It has many advantages over any other methods and most importantly it removes salts, one of the major inhibitors in sequencing (In case of new user facility will train the user for purification). There is no separate cost for bead purification of PCR products. We are giving it free for user(s) who wants to sequence their PCR products.
Labelling of sample and primer tubes:
- If sample or primer is provided in 1.5mL eppendorf tube, then it’s important to use stickers for labelling. This is because the tubes are stored in refrigerated condition which could result in fading of marker dye. Do not submit samples in 0.5mL tube.
- Every eppendorf tube should be label with sample name or number on top and side of the tube as well.
- All sample and primer tubes should have name and date or preferably SS ID in case of less number of samples. If user is submitting sample or primer in 8 well strips at least one tube should have the SS ID. If samples are provided in 96 well plate then please mentioned this ID and name of user on plate. This will help us to differentiate user’s samples even if the same labeling (E.g. A, B, C or 1, 2, 3...etc) is assigned by different users and it helps to trace back your sample(s) and primer(s) as well.
- If samples/primers are assigned with number and in form if the names are provided then it is better to provide small hardcopy of sheet indicating the numbers assigned to corresponding sample(s)/primer(s) or user can mention the same on submission form.
- The sample volume to be submitted is 3µL (minimum) and concentration of sample should be 150ng/µL for plasmid and 10-30ng/µL for purified PCR product. The primer concentration and volume to be provided is 10µM and 5µL (minimum) respectively. If the plasmid is of low copy number the user(s) have to inform the facility.
The samples submitted by user goes through quick quality check (Only 1 or 2 samples from batch!!) and following decisions are made:
- If the concentration is too less for sequencing then, users will be notified for the same. The forms will be edited to zero reaction and user won’t be charged for that reaction(s)
- If sample submitted have high concentration then required, then facility will not dilute the sample for user. It’s user’s responsibility to dilute and submit the sample as required by the facility.
3. Post Sequencing:-
- After successful sequencing, leftover samples and primers will be stored in the facility only for one week. The user may collect the same within the specified time. It is advisable to collect the sample(s)/Primer(s) on same or next day user receives the results.
- If sequencing fails then facility will analyze the issue. During the process facility may ask user to provide certain details about sample(s), primer(s), method of processing sample, overview or summary of experiment or process etc.
- The facility will discuss with user regarding sequencing result via mail or telephone or user will be called to the facility.
- Please note the facility will maintain the confidentiality of user’s data and other then facility no one have access to user’s information. Also the facility requires this information to provide best of knowledge to user and to address the issue majorly at technical point of view. However, facility may provide tentative scientific explanation for improving the quality of sequencing depending on situations.
- The result will be sent to user in 2 working days. However in case of very high sample load the sequencing machine demands maintenance which can result in delays.
- The sequencing for sample will be repeated only if the quality is affected due to sequencer machine. If problem found from user point of view the facility will not repeat the sequencing until user has filled the fresh form.
Sequencing PCR condition:
|Initial denaturation||95 ˚C||2mins|
|S.NO||Primer Name||Primer Sequence 5’-> 3’|
|1||M13 Forward (-21)||5'-TGT AAA ACG ACG GCC AGT-3'|
|2||M13 Forward (-43)||5'-AGG GTT TTC CCA GTC ACG ACG TT-3'|
|3||M13 Forward (-40)||5'-GTTTTCCCAGTCACGAC-3'|
|4||M13 Reverse (-29)||5'-CAG GAA ACA GCT ATG ACC-3'|
|5||M13 Reverse (-49)||5'-GAG CGG ATA ACA ATT TCA CAC AG-3'|
|6||T3||5'-AA TTA ACC CTC ACT AAA GGG-3'|
|7||T7-F version 2.0||5'-GCG CAG TAA TAC GAC TCA CTA TAG G-3'|
|8||T7-Reverse (Terminator)||5'-CT AGT TAT TGC TCA GCG GT-3'|
|9||CMV||5'-CGC AAA TGG GCG GTA GGC GTG-3'|
|10||EGFP-C (Forward)||5'-CAT GGT CCT GCT GGA GTT CGT G-3'|
|11||EGFP-N (Reverse)||5'-CGT CGC CGT CCA GCT CGA CCA G-3'|
|12||U6 Primer||5'-GAG GGC CTA TTT CCCATG ATT CC-3'|
|13||PGEX-6P1-F||5'-GGG CTG GCA AGC CAC GTT TGG TG-3'|
|14||PGEX-6P1-R||5'-GGA GCT GCA TGT GTC AGA GG-3'|
|15||BGH-R||5'-TAG AAG GCA CAG TCG AGG-3'|
|16||SP6||5'- GAT TTA GCT GAC ACT ATA G-3'|