Sam:LabNotes/Microbiome-new/2010-12-29: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Sam Chiang
>Sam Chiang
 
(39 intermediate revisions by 2 users not shown)
Line 1: Line 1:
='''Nextera-library construction on E.coli SAG(WGA1027'10, WGA1103'10)'''=  
='''Nextera-library construction on E.coli SAG(WGA1027'10, WGA1103'10) - test 1'''=  


==Background==
==Background==
Line 26: Line 26:
===EtOH purification of MDA amplicons===
===EtOH purification of MDA amplicons===
*Total volume: 50uL.
*Total volume: 50uL.
**'''UV-decontamination the 100% EtOH, 3M NAOAc and GlycoBlue in crosslinker oven'''for 20min.
**'''UV-decontamination the 100% EtOH, 3M NAOAc and GlycoBlue in crosslinker oven for 20min'''.
**Add 2.5X volume 100% EtOH (125uL) + 0.1X volume 3M NaOAc (5uL) + 1.5uL Glycoblue -> incubate at -80C for 25min.
**Add 2.5X volume 100% EtOH (125uL) + 0.1X volume 3M NaOAc (5uL) + 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.
*'''NOTE:''' White big pellets still showed in short-amplified samples which suggested their appearance has nothing to do with over-dose tRNA adding or the introduced contamination during Covaris shearing (in HMS by Sara). The white pellet may due to MDA regent content, either N6 primers or enzyme proteins.


===Tagmentation reaction===
===Tagmentation reaction===
*Set up tagmentation reaction on cold block:
*Set up tagmentation reaction on cold block:
                           1rxn
                           1rxn   4.5rxn
  DNA(10ng)+ H2O           15.0
  DNA template+H2O         15.0     -
  5X Nextera Rxn buf.(LMW)  4.0
  5X Nextera Rxn buf.(LMW)  4.0  18.0
  Nextera enzyme            1.0
  Nextera enzyme            1.0   4.5
  ------------------------------
  -------------------------------------
                           20.0 uL
                           20.0   22.5/4.5 = 5uL -- template 15uL


*Briefly vortexing and centrifuging. Incubate in thermocycler (using instant incubation) at 55C for 5min.
*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
*During the incubation, setup the tube and buffer of MiniElute columns.
*Reaction products (20uL) were purified using MiniElute column following the standard QIAquick protocol.
**MiniElute columns have lids which are better to avoid introduced contamination.
**The efficiency of MiniElute columns has been verified in Nextera-kit manual.
*Elute DNA in 11uL pre-warmed '''EB buffer'''.


===Zymo DNA purification===
===Bridge-PCR(bPCR) amplification===
*Pre-load 100uL binding buf. in 1.5mL-tubes. Add 20uL reaction product from last step.
*Set up the reaction on cold block in Eppendorf white-bottom PCR strip tubes:
                              1rxn    4.5rxn
RT-PCR grade H2O            16.0    72.0
DNA lib (from last step)      5.0    -
2X Nextera PCR buf.         25.0  112.5
50X Nextera primier cocktail  1.0    4.5 - Illumina-compatible     
50X Nextera adaptor 2        1.0    -  - Using barcoded adaptor (ID1 - ID4)
Nextera PCR enzyme            1.0    4.5
20X Eva-green                1.0    4.5
-----------------------------------------
                              50.0  198.0 uL /4.5= 44 --- 5uL template + 1uL barcoded adaptor
*Eva-green or SYBR-green acceptable range is from 0.2X~0.4X in rt-PCR reaction
*Briefly vortexing and centrifuging.
*Briefly vortexing and centrifuging.
*Transfer the mixture (120uL) onto a Zymo-spin column in a collection tube.
*Cut the tubes from the strip.  
*Centrifuge at 10,000xg for 1min. Discard flowthrough.
*Program "Nextera lib amplification with Nextera-PCR enzyme": 72C 3min-> 95C 30sec -> (95C 10sec -> 62C 30sec -> 72C 3min)x15 cycle -> 4C forever
*Add 250uL washing buf. onto the column. Centrifuge at 10,000xg for 1min. Discard flowthrough.
**Pick up sample around 9 cycles (for 50ng-template reactions).
*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===
==Results==
*Set up the reaction on cold block in PCR tubes:
*Samples were picked from Realplex machine when they arrived about 10000~15000 increasement of fluorescence.
                              1rxn
  [[File:sam122910-qPCR-1.jpg|900px]] '''Monitored fluorescence'''
RT-PCR grade H2O            17.0
  [[File:sam122910-qPCR-3.jpg|900px]] '''Normalized fluorescence'''
DNA lib (from last step)      5.0
  2X Nextera PCR buf.          25.0
50X Nextera primier cocktail  1.0 - Illumina-compatible     
50X Nextera adaptor 2        1.0 - could be stwitch to barcoded library (optional)
Netera PCR enzyme            1.0
  ---------------------------------
                              50.0 uL
*Briefly vortexing and centrifuging.
*Program "Nextera": 72C 3min-> 95C 30sec -> (95C 10sec -> 62C 30sec -> 72C 3min)x10 cycle -> 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).


==Ampure bead purification==
  Sample      NX1    NX2  NX3  NX4
*Following standard protocol with 0.7V volume AMpure beads.
              ----------------------
*Nanodrop the purified DNA and stored it in -20C immediately.
  Well        A1          A3     
                    B2          B4
  Min        33    29    58    29
  Cycle        7      6    13    6
  Dif. Fluorescence(monitored)
          12000  8000 11000  8000 
 
*'''The starting fluorescent intensities for each of templates are consistent with their estimated DNA concentration (by QUBIT-HS)''', which suggested that the QUBIT-HS quantification is relatively accurate.  
Library    DNA starting amount    qPCR starting fluorescence(monitored)
NX1        288ng                  10000
NX2        500ng                  17000
NX3        114ng                  4000
NX4        500ng                  16000


==Results==
==Libraries validation / quantification==
*3uL of amplified product was validated using TBE-PAGE  
*Amplified libraries were validated by TBE-PAGE. Ref. link: http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-3-2
**3uL PCR product + 3uL 0.5X TBE + 3uL 6X loading dye.
**3uL PCR products were run on 10-well PAGE (200V, 30min).
  [[File:sam072410-Nextera lib.jpg|250px]]
**Sample well: 1uL sample + 3uL 6X loading dye + 6uL 0.5X TBE buffer
*I accidently over-run the gel (250V, 30min). I should only use 200V instead.
**Ladder well: 1uL low mass ladder + 3uL 6X loading dye + 6uL 0.5X TBE buffer
*The bands showed up but look smear.
***Load 6uL ladder on the 1st well and 3uL on the 2nd well
**'''For the 10ng starting amount, 10cycle PCR amplification is not enough.
**Use 3uL SYBR-gold with regular staining(10min) with foil protection
  [[File:sam122910 lib-3.jpg|350px]]  [[File:sam122910 lib-quant-2.jpg|340px]]
*Quantification of library concentration
**Ref. link: http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-11-14
*The quantification file [[File:sam122910-quantification.xls]]
[[File:sam122910-regression line.jpg|400px]]
                  Total yield(ng)
NX1 2.51 ng/uL  125.5 ng
NX2 1.27 ng/uL    63.5 ng
NX3 0.67 ng/uL    33.5 ng
NX4 0.30 ng/uL    15.0 ng


='''Extra-library amplification using rt-PCR'''=
==Discussion==
*Since there are 10uL template left from the 1st step of Nextera library construction. Only 5uL were used for PCR amplification.
*Nextera kit can work up to 500ng template.
*I am trying to use the other 5uL template(transposon tagged DNA) for amplification.
*Nextera kit works better with 50-100ng template. '''Too much tempalte (300ng-500ng) will cause the problem on tagmentation reaction(using LMW buffer)'''. The library DNA will not be distributed with 200~400bp properly.
*Set up the reaction on cold block in EP-white PCR-strip tubes:
*It is unclear why library NX3 needs to be amplified to the 13th cycle. However it showed the best size distribution within 200~400bp.
                              1rxn    x2.2 rxn
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 19:56, 2 May 2012

Nextera-library construction on E.coli SAG(WGA1027'10, WGA1103'10) - test 1[edit]

Background[edit]

Sample preparation[edit]

Trial-1(12-29-'10)

 Sample no.   Sample name          Conc.(ng/uL)  Sample(uL)      tRNA("2")   H2O    Total DNA(ng)
 ---------------------------------------------------------------------------------------------------
 NX01         WGA102710-(1-4)- 3hr     5.77         50.00           0.0       0.00   288.50ng
 NX02         WGA102710-(1-4)-10hr  1162.79          0.43            -       49.00   500.00ng
 NX03         WGA110310-(5)-   3hr     2.29         50.00           0.0       0.00   114.50ng
 NX04         WGA110310-(5)-  10hr   555.81          0.90            -       49.00   500.00ng
  • I am trying not to added any tRNA carrier in for EtOH precipitation since the samples amount for short-amplification is around 100~300ng.

EtOH purification of MDA amplicons[edit]

  • Total volume: 50uL.
    • UV-decontamination the 100% EtOH, 3M NAOAc and GlycoBlue in crosslinker oven for 20min.
    • Add 2.5X volume 100% EtOH (125uL) + 0.1X volume 3M NaOAc (5uL) + 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.
  • NOTE: White big pellets still showed in short-amplified samples which suggested their appearance has nothing to do with over-dose tRNA adding or the introduced contamination during Covaris shearing (in HMS by Sara). The white pellet may due to MDA regent content, either N6 primers or enzyme proteins.

Tagmentation reaction[edit]

  • Set up tagmentation reaction on cold block:
                          1rxn   4.5rxn
DNA template+H2O          15.0     -
5X Nextera Rxn buf.(LMW)   4.0   18.0
Nextera enzyme             1.0    4.5
-------------------------------------
                          20.0   22.5/4.5 = 5uL -- template 15uL
  • Briefly vortexing and centrifuging. Incubate in thermocycler (using instant incubation) at 55C for 5min.
  • During the incubation, setup the tube and buffer of MiniElute columns.
  • Reaction products (20uL) were purified using MiniElute column following the standard QIAquick protocol.
    • MiniElute columns have lids which are better to avoid introduced contamination.
    • The efficiency of MiniElute columns has been verified in Nextera-kit manual.
  • Elute DNA in 11uL pre-warmed EB buffer.

Bridge-PCR(bPCR) amplification[edit]

  • Set up the reaction on cold block in Eppendorf white-bottom PCR strip tubes:
                             1rxn    4.5rxn
RT-PCR grade H2O             16.0    72.0
DNA lib (from last step)      5.0     -
2X Nextera PCR buf.          25.0   112.5 
50X Nextera primier cocktail  1.0     4.5 - Illumina-compatible       
50X Nextera adaptor 2         1.0     -   - Using barcoded adaptor (ID1 - ID4)
Nextera PCR enzyme            1.0     4.5
20X Eva-green                 1.0     4.5
-----------------------------------------
                             50.0   198.0 uL /4.5= 44 --- 5uL template + 1uL barcoded adaptor
*Eva-green or SYBR-green acceptable range is from 0.2X~0.4X in rt-PCR reaction
  • Briefly vortexing and centrifuging.
  • Cut the tubes from the strip.
  • Program "Nextera lib amplification with Nextera-PCR enzyme": 72C 3min-> 95C 30sec -> (95C 10sec -> 62C 30sec -> 72C 3min)x15 cycle -> 4C forever
    • Pick up sample around 9 cycles (for 50ng-template reactions).

Results[edit]

  • Samples were picked from Realplex machine when they arrived about 10000~15000 increasement of fluorescence.
File:Sam122910-qPCR-1.jpg Monitored fluorescence
File:Sam122910-qPCR-3.jpg Normalized fluorescence
 Sample      NX1    NX2   NX3   NX4
             ----------------------
 Well        A1           A3       
                    B2          B4 
 Min         33     29    58    29 
 Cycle        7      6    13     6 
 Dif. Fluorescence(monitored) 
          12000   8000 11000  8000  
  
  • The starting fluorescent intensities for each of templates are consistent with their estimated DNA concentration (by QUBIT-HS), which suggested that the QUBIT-HS quantification is relatively accurate.
Library    DNA starting amount    qPCR starting fluorescence(monitored)
NX1        288ng                  10000 
NX2        500ng                  17000
NX3        114ng                   4000
NX4        500ng                  16000

Libraries validation / quantification[edit]

  • Amplified libraries were validated by TBE-PAGE. Ref. link: http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-3-2
    • 3uL PCR products were run on 10-well PAGE (200V, 30min).
    • Sample well: 1uL sample + 3uL 6X loading dye + 6uL 0.5X TBE buffer
    • Ladder well: 1uL low mass ladder + 3uL 6X loading dye + 6uL 0.5X TBE buffer
      • Load 6uL ladder on the 1st well and 3uL on the 2nd well
    • Use 3uL SYBR-gold with regular staining(10min) with foil protection
File:Sam122910 lib-3.jpg  File:Sam122910 lib-quant-2.jpg
File:Sam122910-regression line.jpg
                 Total yield(ng)
NX1 2.51 ng/uL   125.5 ng
NX2 1.27 ng/uL    63.5 ng
NX3 0.67 ng/uL    33.5 ng
NX4 0.30 ng/uL    15.0 ng

Discussion[edit]

  • Nextera kit can work up to 500ng template.
  • Nextera kit works better with 50-100ng template. Too much tempalte (300ng-500ng) will cause the problem on tagmentation reaction(using LMW buffer). The library DNA will not be distributed with 200~400bp properly.
  • It is unclear why library NX3 needs to be amplified to the 13th cycle. However it showed the best size distribution within 200~400bp.