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.