- First you must PCR amplify your pool to add new "A" at the end by Taq polymerase. Just run the pool with 10 cycles under normal conditions.
- Follow the general TA cloning kit instructions by Invitrogen to ligate your template into the pCR 2.1 vector with T4 ligase. Clone the ligated plasmid vector into Top10 cells and plate onto LB+Amp plates with X-gal.
- Clone check at least 11 white colonies/plate with M13F/R primers. I typically do multiples of 8 or 12 and run 7 or 11 down a gel with a ladder. Try to set up volume transfers to use multichannel pipettors where appropriate.
- Pick the colony with a pipette tip, shake in 1.5ml liquid LB + Amp then add to a 25ul PCR reaction - Grow overnight
- Run the PCR at 50C annealing for 25 cycles.
- Run down a gel and analize the correct size bands
- (for our pools it will be ~200bp - incorrect, ~300bp - correct)
- Clean up the PCR reactions with a general silica filter column PCR cleanup kit (2-24 reactions fit easily into the centrifuges) or with a Millipore MultiScreen PCRµ96 plates (cat# LSKMPCR10) in the Ellington lab (>24 reactions).
- Add the PCR reactions to unused wells.
- Put the plate on a Qiagen QiaVac96 & use a water pull vacuum to remove salts & sol’n from reactions. Pull vacuum until wells are dry.
- Elute clean PCR reactions by adding 50 uL of water to the plate, shake plate for 5-10 minutes at room temp, pipet up & down to resuspend products, then holding the plate at an angle transfer the solution above the filter into a clean labeled tube.
- Make a 1:10 dilution of your reactions (brings most down to ~10ng/ul and spec if necessary).
- In a 1.5ml tube, add 1ul of diluted template (10ng total) + 1ul of 10uM primer (10pmol total) + 10ul diH2O
- Fill in coreweb.icmb.utexas.edu form and take over to the core.
- You only use one primer per sequencing reaction. Feel free to you use core primers but choose the one specific to the pCR 2.1 vector:
- M13R (-24) 5'-GGAAACAGCTATGACCATG-3'
- The T7 sequencing Primer (forward) 5'-AAATTAACCCTCACTAAAGG-3'
- The M13 Universal Primer (-20) Forward 5'-GTAAAACGACGGCCAGT-3'
- Purify the plasmids from the liquid cultures that proved successful for long term storage using a mini plasmid prep kit. You can also spin the cells down, remove the supernatant and freeze the cells at -80C till the sequencing results return.
Notes:
- I've found that sequencing cloned pool PCR products (~300bp) works very well when not too concentrated. I've actually had success from 1ng to 80ng template in the 12ul total reaction when the template is very small. Generally 1-10ng works great. Others have gone as low as 0.1ng target with 3pmol primer with success when higher concentrations failed. Above 50ng does not work well for PCR reactions for me. Cecil in the core recommends 5ng/100bp so 15ng/300bp fragments.
- Primer quantities can be as low as 3pmol total.
- The pCR 2.1 vector is 3.9kb and you must add your insert to the length when submitting plasmid for sequencing. A copy of the plasmid map can be found here.
- Plasmid templates for sequencing work very well also, but extra time is involved waiting for the culture to grow overnight. When using plasmid template, 500ng total template is required per 12ul sequencing reaction. This is typically a 2-5 ul of the plasmid prep.
If the UT core facility does not work well for your targets, it could be their fault or it could be the cleanliness of your template. Here is an exhaustive link of many different sequencing houses: http://www.nucleics.com/DNA_sequencing_support/sequencing-service-reviews.html
In Houston, SeqWrite and Lone Star Labs are two notable labs.
