Sam:LabNotes/Microbiome-new/2010-8-4: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Sam Chiang
No edit summary
>Zsakura2
 
(17 intermediate revisions by one other user not shown)
Line 10: Line 10:
*Four samples will be made Nextera libraries with barcoded primers.
*Four samples will be made Nextera libraries with barcoded primers.


*I also made extention-MDA amplicon for this sample (additinoal 8hr amplification)
==Sample preparation==
*These two samples will be used for making sequencing libraries:
Name    DNA(20ng)  tRNA carrier(480ng)   RT-PCR H2O
**Sam070610-90m-C (0.3ng/uL, 50uL)
----------------------------------------------------
**Sam070610-90m-C-ext (379ng/uL, 20uL)
90m-3  45.8        4.8                  49.4
10h-3    2.0       4.8                  93.2
90m-4  40.0        4.8                  55.2
10h-4    2.3        4.8                  92.9


==Sample preparation==
*Only use about 20ng DNA for starting amount (10ng is the total available amount for the sample Sam072710-90m-3)
*Only use about 10ng DNA for starting amount (10ng is the total available amount for the sample Sam070610-90m-C)
**Use all of remained 30uL for Sam070610-90m-C (0.3ng/uL, 50uL)=> (~10ng)
**Make 1/100 dilution for Sam070610-90m-C-ext(379ng/uL, 20uL)=> 3.8ng/uL => Use 3uL of this diluted template (~10ng)




===EtOH purification of MDA amplicons===
===EtOH purification of MDA amplicons===
**Add 4.9uL tRNA carrier(100ng/uL) into MDA amplicon.
*Add 4.8uL tRNA carrier(100ng/uL) into MDA amplicon.
**Add H2O to make total valume to 100uL.
*Add H2O to make total valume to 100uL.
**Add 2.5X volume 100% EtOH (250uL) + 0.1X volume 3M NaOAc (10uL) + 1.5uL Glycoblue -> incubate at -80C for 25min.
*Add 2.5X volume 100% EtOH (250uL) + 0.1X volume 3M NaOAc (10uL) + 1.5uL Glycoblue -> incubate at -80C for 25min.
**Centrifuge at 10,000xg, 4C for 25min -> resuspend the pellet with 750uL 75% EtOH.
*Centrifuge at 10,000xg, 4C for 25min -> resuspend the pellet with 750uL 75% EtOH.
**Centrifuge at 10,000xg, 4C for 5min. Elute the pellet in 15uL RT-PCR grade H2O.
*Centrifuge at 10,000xg, 4C for 5min. Elute the pellet in 15uL RT-PCR grade H2O.


===Tagmentation reaction===
===Tagmentation reaction===
Line 45: Line 45:
*Transfer the mixture (120uL) onto a Zymo-spin column in a collection tube.
*Transfer the mixture (120uL) onto a Zymo-spin column in a collection tube.
*Centrifuge at 10,000xg for 1min. Discard flowthrough.
*Centrifuge at 10,000xg for 1min. Discard flowthrough.
*Add 250uL washing buf. onto the column. Centrifuge at 10,000xg for 1min. Discard flowthrough.
*Add 250uL washing buf. onto the column. Centrifuge at 10,000xg for 1min. Discard flow-through.
*Repeat the last step again.
*Repeat the last step again.
*Perform an additional centrifuge at 10,000xg for 1min (to eliminate any residue).
*Perform an additional centrifuge at 10,000xg for 1min (to eliminate any residue).
Line 52: Line 52:


===Library enrichment by PCR amplification===
===Library enrichment by PCR amplification===
*Set up the reaction on cold block in PCR tubes:
*In order to use the libraries for cloning sequencing, we have to switch to the Phusion enzyme instead.
**The Phusion enzyme provide blunt end PCR products which is suitable for blunting cloning.
**We also need to add Isotherm (Epicentre) enzyme to repair the gaps left by transposon taggmentation.
 
*In order to test the new amplification recipe, I only tried two of four samples with barcoded(Index) primer
**90m-4(IDX1) and 10hr-4(IDX2)
*Set up the qPCR reaction (50uL) on cold block in PCR tubes:
                               1rxn
                               1rxn
  RT-PCR grade H2O            17.0
  RT-PCR grade H2O            17.1
  DNA lib (from last step)      5.0
  DNA lib (from last step)      5.0
  2X Nextera PCR buf.          25.0
  2X Phusion master mix        25.0
  50X Nextera primier cocktail  1.0 - Illumina-compatible       
  50X Nextera primier cocktail  1.0 - Illumina-compatible       
  50X Nextera adaptor 2        1.0 - could be stwitch to barcoded library (optional)
  50X Nextera adaptor 2        1.0 - Using IDX1 and IDX2
  Netera PCR enzyme            1.0
  50X SYBR I                    0.4
Isothermo(Epicentre)          0.5
  ---------------------------------
  ---------------------------------
                               50.0 uL
                               50.0 uL
*Briefly vortexing and centrifuging.
*Briefly vortexing and centrifuging.
*Program "Nextera": 72C 3min-> 95C 30sec -> (95C 10sec -> 62C 30sec -> 72C 3min)x10 cycle -> 4C forever
**65C 3min -> 98C 30sec -> (98C 5sec -> 65C 15sec -> 72C 20sec)x15 cycle -> 72C 3min -> 4C forever'''
*'''NOTE: The manual suggested only amplify 9 cycle. Since my starting amount is only 10ng (instead of 50ng), I amplify one more cycle (total 10cycle).
**'''NOTE1.: This program is adapted from Dr.Zhang's protocol 0805'10. The temperature setting is different from original Nextera protocol.
**'''NOTE2.: The 65C 3min is for Isothermo enzyme. The 98C 30sec is for Phusion (Hot-start) enzyme'''.
*Stop the reaction before the amplification reach saturation.
**Sample 90m-4 stopped at 16cycle
**Sample 10h-4 stopped at 8cycle


==Ampure bead purification==
==Ampure bead purification==
*Following standard protocol with 0.7V volume AMpure beads.
*Following standard protocol with 0.7V volume AMpure beads.
*Nanodrop the purified DNA and stored it in -20C immediately.
*Nanodrop the purified DNA(optional) and stored it in -20C immediately.


==Results==
==Results==
*3uL of amplified product was validated using TBE-PAGE  
*3uL of amplified product was validated using TBE-PAGE  
*3uL amplified libraries were validated using 6%TBE-PAGE with standard long staining method(250V, 20min).
**3uL WGA amplicon + 3uL 0.5X TBE + 3uL 6X loading dye.
**3uL WGA amplicon + 3uL 0.5X TBE + 3uL 6X loading dye.
[[File:sam072410-Nextera lib.jpg|250px]]
*I accidently over-run the gel (250V, 30min). I should only use 200V instead.
*The bands showed up but look smear.
**'''For the 10ng starting amount, 10cycle PCR amplification is not enough.


[[File:sam080510- Nextera lib amp - phusion EZ.jpg|600px]] [[File:sam080610- Nextera-lib amplified.jpg|300px]]


='''Extra-library amplification using rt-PCR'''=
*The templates from these qPCR amplification is 10ng
*Since there are 10uL template left from the 1st step of Nextera library construction. Only 5uL were used for PCR amplification.
**Start with 20ng but only 1/2 of 1-step product were amplified.
*I am trying to use the other 5uL template(transposon tagged DNA) for amplification.
**The sample 10h-4 may contain more template than 90m-4 so it was amplified faster.
*Set up the reaction on cold block in EP-white PCR-strip tubes:
**The sample 10h-4 was prepared by dilution method to get estimated 20ng as starting amount. It is possible that the inaccuracy of quantification cause higher template used in 10h-4 sample.
                              1rxn    x2.2 rxn
*Using 10ng templates for qPCR is not stable in this method, even with 16cycle amplification the PCR amplicons still hard to be seen. However if I keep amplifying the background may start showing up.
RT-PCR grade H2O            16.8    36.96
DNA lib (from last step)      5.0      -
2X Nextera PCR buf.          25.0    55.00
50X Nextera primier cocktail  1.0      2.20  - Illumina-compatible     
50X Nextera adaptor 2        1.0      2.20  - could be stwitch to barcoded library (optional)
Netera PCR enzyme            1.0      2.20 
50X SYBR-green I              0.2      0.44
-------------------------------------------
                              50.0    99.00/2.2=45 --- template 5uL
'''NOTE: I use the "no.2" SYBR-green 50X'''
Stop the reaction before the amplification curve reach pleatue
 
==Results==
*3uL amplified libraries were validated using 6%TBE-PAGE with standard long staining method(200V, 30min).
[[File:sam072910-nextera lib amp-2nd amp.jpg|300px]]
*The 90min-library showed good range between 200-400bp
*The 10hr-library is probably over-loaded on gel. I need to do the nanodrop on this amplicon and validate again.
**After AMpure amplification the concentration could be raised up becasued the totoal volume was changed from 50uL to 30uL (I used 30uL H2O to elute the purified DNA).
*The comparison of before and after AMpure bead purification showed no significatn loss with the purification method.

Latest revision as of 23:36, 29 December 2010

Nextera-library construction on single E.coli MDA amplicons(0727'10 MDA)[edit]

Background[edit]

  • The positive amplicons are:
    • Well-3(E)-10hr-sam072710, well-4(F)-10hr-sam072710
    • I did the QUBIT-HS for both amplcons and their original 90m amplicons.
File:Sam080410 - QUBIT -new.jpg
  • Four samples will be made Nextera libraries with barcoded primers.

Sample preparation[edit]

Name    DNA(20ng)   tRNA carrier(480ng)   RT-PCR H2O
----------------------------------------------------
90m-3   45.8        4.8                   49.4
10h-3    2.0        4.8                   93.2
90m-4   40.0        4.8                   55.2
10h-4    2.3        4.8                   92.9
  • Only use about 20ng DNA for starting amount (10ng is the total available amount for the sample Sam072710-90m-3)


EtOH purification of MDA amplicons[edit]

  • Add 4.8uL tRNA carrier(100ng/uL) into MDA amplicon.
  • Add H2O to make total valume to 100uL.
  • Add 2.5X volume 100% EtOH (250uL) + 0.1X volume 3M NaOAc (10uL) + 1.5uL Glycoblue -> incubate at -80C for 25min.
  • Centrifuge at 10,000xg, 4C for 25min -> resuspend the pellet with 750uL 75% EtOH.
  • Centrifuge at 10,000xg, 4C for 5min. Elute the pellet in 15uL RT-PCR grade H2O.

Tagmentation reaction[edit]

  • Set up tagmentation reaction on cold block:
                          1rxn
DNA(10ng)+ H2O            15.0
5X Nextera Rxn buf.(LMW)   4.0
Nextera enzyme             1.0
------------------------------
                          20.0 uL
  • Briefly vortexing and centrifuging. Incubate in thermocycler (using instant incubation) at 55C for 5min.
  • During the incubation, setup the tube and buffer of Zymo DNA cleaning kit

Zymo DNA purification[edit]

  • Pre-load 100uL binding buf. in 1.5mL-tubes. Add 20uL reaction product from last step.
  • Briefly vortexing and centrifuging.
  • Transfer the mixture (120uL) onto a Zymo-spin column in a collection tube.
  • Centrifuge at 10,000xg for 1min. Discard flowthrough.
  • Add 250uL washing buf. onto the column. Centrifuge at 10,000xg for 1min. Discard flow-through.
  • Repeat the last step again.
  • Perform an additional centrifuge at 10,000xg for 1min (to eliminate any residue).
  • Add 11uL pre-warmed H2O onto the column, transfer the column onto a 1.5mL-tube. Incubate at RT for 2min.
  • Centrifuge at 10,000xg for 1min. The elute DNA is about 10~11uL. Only use 5uL for the library amplification. (Label and store the other 5uL product in -20C)

Library enrichment by PCR amplification[edit]

  • In order to use the libraries for cloning sequencing, we have to switch to the Phusion enzyme instead.
    • The Phusion enzyme provide blunt end PCR products which is suitable for blunting cloning.
    • We also need to add Isotherm (Epicentre) enzyme to repair the gaps left by transposon taggmentation.
  • In order to test the new amplification recipe, I only tried two of four samples with barcoded(Index) primer
    • 90m-4(IDX1) and 10hr-4(IDX2)
  • Set up the qPCR reaction (50uL) on cold block in PCR tubes:
                             1rxn
RT-PCR grade H2O             17.1
DNA lib (from last step)      5.0
2X Phusion master mix        25.0
50X Nextera primier cocktail  1.0 - Illumina-compatible       
50X Nextera adaptor 2         1.0 - Using IDX1 and IDX2 
50X SYBR I                    0.4
Isothermo(Epicentre)          0.5
---------------------------------
                             50.0 uL
  • Briefly vortexing and centrifuging.
    • 65C 3min -> 98C 30sec -> (98C 5sec -> 65C 15sec -> 72C 20sec)x15 cycle -> 72C 3min -> 4C forever
    • NOTE1.: This program is adapted from Dr.Zhang's protocol 0805'10. The temperature setting is different from original Nextera protocol.
    • NOTE2.: The 65C 3min is for Isothermo enzyme. The 98C 30sec is for Phusion (Hot-start) enzyme.
  • Stop the reaction before the amplification reach saturation.
    • Sample 90m-4 stopped at 16cycle
    • Sample 10h-4 stopped at 8cycle

Ampure bead purification[edit]

  • Following standard protocol with 0.7V volume AMpure beads.
  • Nanodrop the purified DNA(optional) and stored it in -20C immediately.

Results[edit]

  • 3uL of amplified product was validated using TBE-PAGE
  • 3uL amplified libraries were validated using 6%TBE-PAGE with standard long staining method(250V, 20min).
    • 3uL WGA amplicon + 3uL 0.5X TBE + 3uL 6X loading dye.
File:Sam080510- Nextera lib amp - phusion EZ.jpg File:Sam080610- Nextera-lib amplified.jpg
  • The templates from these qPCR amplification is 10ng
    • Start with 20ng but only 1/2 of 1-step product were amplified.
    • The sample 10h-4 may contain more template than 90m-4 so it was amplified faster.
    • The sample 10h-4 was prepared by dilution method to get estimated 20ng as starting amount. It is possible that the inaccuracy of quantification cause higher template used in 10h-4 sample.
  • Using 10ng templates for qPCR is not stable in this method, even with 16cycle amplification the PCR amplicons still hard to be seen. However if I keep amplifying the background may start showing up.