Sam:LabNotes/Microbiome-new/2010-4-16
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Sanger sequencing troble shooting - mp1-mp8 amplicons (PCR products from 04-04-'10)[edit]
Background[edit]
- The the 2nd Sanger seqeuencing was performed with special "very-short PCR primer"(The primer modifie from regular library amplification primer with no extension tail and only 21nt => Tm=59).
- The Sanger sequencing results still showed a very low sequence quality due to the unspecific amplicons.
Possible reasons and improvements (Discussed with technical support):[edit]
- There are unspecific amplification. Unspecific amplication were difficul to be seen and removed by E-gel and still could be amplified in the 2nd round PCR.
- Sol.1 => Use the old school trick to do gel cutting on target band for sequencing.
- Sol.2 => Increase the annealing temperature. Choose the good annealing temp by doing a gradient PCR.
- The original templates (insert) have contaminations.
- The original inserts of these libraries is PCR product, probably the inserts originally contain the contamination.
- Since I am using the primers that can bind on adaptors, any thing ligated by the adaptor could be amplified.
- I assume that Sanger sequencing using Lib. amplification primer is more difficult then using gene specific primers, in terms of getting good quality of sequencing results.
Optimizing annealing tempurature[edit]
- Test possible annealing temputrature beteen 50C~70C using gradient PCR program (Program: G-PCR) - program set up (07-01-2009)
1rxn 8.5rxn x 2 H2O 10.0 85.0 DNA 0.5 4.25 Primer-f(10uM) 1.0 8.50 ----- "short-PCR primer" / "very-short PCR primer" Primer-r(10uM) 1.0 8.50 ----- "short-PCR primer" / "very-short PCR primer" 2X Fusion 12.5 105.25 ----------------------------- 25.0 212.5 / 8.5=25
Results[edit]
- PCR program (G-PCR):
Step1 94C 2min Step2 94C 40sec Step3 50C-70C 40sec Step4 72C 1min Step5 Goto step2 for 34 cycles Step6 72C 10min Step7 End
File:Sam041610-gradient PCR on short -PCR primer.jpg File:Sam041610-gradient PCR on very-short -PCR primer.jpg C.3~C.10 (column 3 ~ column 10)
- The expected (designed) Annealing temp:
- short-PCR primeris 64C
- very-short-PCR primer is 59C
- The results suggest that not obvious difference in product using different Annealing temperature.
- I will just keep using the original Tm for these two primers.
Sample preparation for new Sanger seqneucneing[edit]
Large volume amplification (continued on 04-23-'10)[edit]
- Amplify the original mPE1 - mPE8 libraries (04-03-'10) into 50uL in order to get enough amount for Gel-cut recovery.
- Add one more reaction for NTC (H2O control)
1rxn 9.5rxn H2O 20.0 190.0 DNA 1.0 - --- from the oiginal PCR amplified mPE1 - mPE8 libraries Primer-f(10uM) 2.0 19.0 --- "very-short PCR primer" Primer-r(10uM) 2.0 19.0 --- "very-short PCR primer" 2X Econo 25.0 237.5 ----------------------------- 50.0 465.5 / 9.5= 49.0 --- Template 1uL
- PCR program "59T20C"
- 94C 2min -> (94C 40sec -> 59C 40sec-> 72C 40sec)x20cycle -> 72C 10min -> 15C forever
Gel electrophoresis and gel-cut[edit]
- 10uL 6X loading dye was added into each reaction (total volume will be 10+50=60uL). Mix by vortexing followed by quick spinning.
- Load 30uL samples into wells of 2% agarose gel made with 14-well comb using the long beak tips.
- Run the gel with 135V for 25min.
File:Sam042310-PCR products in big volume.jpg File:Sam042310-PCR products in big volume-post cut.jpg
- The cut gel slice (~100uL)was placed in 1.5-mL tube. Add 300uL ADB solution into tubes to dissolve the agarose gel.
- Incubat on 55C hotplate for 10min. Briefly vortexing to mix the dissolved gel.
- Transfer the melted agarose mixture onto Zymo-spin column in a collection tube. Centrifuge at 10,000xg for 30sec. Discard the flow-through
- Add 200uL wash buffer (added EtOH into wash buffer first). Centrifuge at 10,000xg for 30sec. Discard the flow-through.
- Repeat the washing step again.
- Add 10uL clean water on the filter. Incubate at RT for 1min. Transfet the filter on a clean 1.5-mL tubes. Centrifuge at 10,000xg for 1min to elute the DNA.
- Nanodrop the eluted DNA.
Results[edit]
File:Sam042310-nanodrop for mp1-mp8 by zymo.jpg
- The recovered concentration is around 20~40ng/uL in 10uL.