I have already amplified rbcl, matk, trnl-f, psba-trnh and ITS regions of my study plants. Now I will be doing sequencing of those regions. I have purified those PCR products by Quaquick gel extraction kit. Now what are the steps, I have to follow before loading onto sequencing?? We have BigDye terminator Kit
Can anybody kindly tell me the detailed steps of cycle sequencing and other procedure?
After the pcr, there are three steps before proceeding to DNA Sequencing:
I) PCR cleanup
Removal of primer dimers you have to go for ExoSAP (Exo is exonuclease I and SAP is shrimp alkaline phospatase cleanup, here is protocol for it:
Reaction vol. 4.25 ul: SAP - 1 ul, Exo I - 0.5 ul, SAP 10x - 2 ul and PCR product - 2.5 ul.
Incubation: 37 °C for 45 minutes (this degrades your primer dimers), 80 °C for 15 minutes( this inactivates your EXoSAP, so that it would't interfers during sequencing reaction)
II) Cycle Sequencing (one way pcr): These amplicons were forwarded for cycle sequencing reaction using BigDye® Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems, Inc.) with 16th fold dilution.
Reaction vol. 10 ul: Ready reaction mix- 0.5 ul, Dilution buffer- 1.75ul, Primers- 2ul, MilliQ water (ddH2O) - 4.75, Template- 1 ul.
(Note: primers for this reaction were used those used in PCR, two sets were prepared one for Forward primer and other for Reverse primer thus said as one way pcr)
Thermal cycler protocol: The thermal cycler conditions were an initial 5 step of 2 min at 96⁰C and 35 cycles of 30 sec at 96⁰C, 15 sec at 55⁰C, and 4 min at 60⁰C.
III) Sequencing Cleanup: This is usually done with ethanol precipitation
1) Set centrifute to 4 °C, 2) mix 7.5M Ammonium acetate with 100% ethanol and vortex (for half reac. 1uL Amm. Ac./sample, 27.5 μL EtOH/sample), 3) Add 28.5 μL of mix from step 2 to each cycle sequencing half-reaction and vortex. 4) Centrifuge samples @ about 4000 rpm for 30 min at 4 °C. (Don’t let samples sit for more than 5 min after spin or DNA may resuspend). 5) With ten minutes left in spin, turn centrifuge temperature back to 25 °C. 6) Take out samples, remove lids, and cover openings with several paper towels folded in half. Gently invert samples and shake. DO NOT BANG PLATE ON THE COUNTER!!! Check tubes and repeat if necessary. 7) Get new dry and clean paper towels and load towels and samples inverted into centrifuge. Spin @ 600 rpm for 3 min. (watch rpm!!!) 8) After spin, check that samples are dry. If not, spin again. (You want to get out all EtOH with unicorporated dye terminators.) 9) When samples are dry, add 200 μL 70% EtOH to each sample, put on lids, and spin @ 4000rpm for 5 min at 25 °C. 10) Repeat step 6. 11) Do inverted spin @ 600 rpm for 5 min at 25 °C. Make sure most of the EtOH is gone. Repeat short spin if necessary. 12) Place under hood to dry for 15-20 minutes. 13) Resuspend in 5 μL loading solution for half-reactions, vortex, spin down, and put in –20 °C freezer until ready to run. (If you plan to store your samples for a while, we recommend storing them dried down and resuspending closer to your sequencing run because formamide will eventually degrade DNA)
Do the sequencing PCR using the same primers (which were used for amplification of respective genes/ DNA SEQUENCES), purify the sequencing PCR products , go for sequencing by adding required chemicals.
After the pcr, there are three steps before proceeding to DNA Sequencing:
I) PCR cleanup
Removal of primer dimers you have to go for ExoSAP (Exo is exonuclease I and SAP is shrimp alkaline phospatase cleanup, here is protocol for it:
Reaction vol. 4.25 ul: SAP - 1 ul, Exo I - 0.5 ul, SAP 10x - 2 ul and PCR product - 2.5 ul.
Incubation: 37 °C for 45 minutes (this degrades your primer dimers), 80 °C for 15 minutes( this inactivates your EXoSAP, so that it would't interfers during sequencing reaction)
II) Cycle Sequencing (one way pcr): These amplicons were forwarded for cycle sequencing reaction using BigDye® Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems, Inc.) with 16th fold dilution.
Reaction vol. 10 ul: Ready reaction mix- 0.5 ul, Dilution buffer- 1.75ul, Primers- 2ul, MilliQ water (ddH2O) - 4.75, Template- 1 ul.
(Note: primers for this reaction were used those used in PCR, two sets were prepared one for Forward primer and other for Reverse primer thus said as one way pcr)
Thermal cycler protocol: The thermal cycler conditions were an initial 5 step of 2 min at 96⁰C and 35 cycles of 30 sec at 96⁰C, 15 sec at 55⁰C, and 4 min at 60⁰C.
III) Sequencing Cleanup: This is usually done with ethanol precipitation
1) Set centrifute to 4 °C, 2) mix 7.5M Ammonium acetate with 100% ethanol and vortex (for half reac. 1uL Amm. Ac./sample, 27.5 μL EtOH/sample), 3) Add 28.5 μL of mix from step 2 to each cycle sequencing half-reaction and vortex. 4) Centrifuge samples @ about 4000 rpm for 30 min at 4 °C. (Don’t let samples sit for more than 5 min after spin or DNA may resuspend). 5) With ten minutes left in spin, turn centrifuge temperature back to 25 °C. 6) Take out samples, remove lids, and cover openings with several paper towels folded in half. Gently invert samples and shake. DO NOT BANG PLATE ON THE COUNTER!!! Check tubes and repeat if necessary. 7) Get new dry and clean paper towels and load towels and samples inverted into centrifuge. Spin @ 600 rpm for 3 min. (watch rpm!!!) 8) After spin, check that samples are dry. If not, spin again. (You want to get out all EtOH with unicorporated dye terminators.) 9) When samples are dry, add 200 μL 70% EtOH to each sample, put on lids, and spin @ 4000rpm for 5 min at 25 °C. 10) Repeat step 6. 11) Do inverted spin @ 600 rpm for 5 min at 25 °C. Make sure most of the EtOH is gone. Repeat short spin if necessary. 12) Place under hood to dry for 15-20 minutes. 13) Resuspend in 5 μL loading solution for half-reactions, vortex, spin down, and put in –20 °C freezer until ready to run. (If you plan to store your samples for a while, we recommend storing them dried down and resuspending closer to your sequencing run because formamide will eventually degrade DNA)
I agreed with Artur Burzynski. You can sent your samples to MACROGEN Corp. or another corporation to perform the purification and sequencing (10dlls with the purification per sample).
- Badges
- Science method