Brandon:LabNotes/Project1/2013-6-10: Difference between revisions

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| 9. 1 uL elute 6. NTC (no tspn) T2||33.8997552||12652.46038||10||338.997552||N/A||
| 9. 1 uL elute 6. NTC (no tspn) T2||33.8997552||12652.46038||10||338.997552||N/A||
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===conclusions===
*will now add 30 uL of water was added to samples, and then 100 uL of beads for a total of 150 ul in solution. Rest of the beads purification protocol will be followed as normal, with ~3 minutes used for drying the beads.
*will process this sample through to test replicates, to see if the polyA issue solved.
*can do one more trial of beads purification, increasing ratio of beads to sample again to ensure everything is working correctly.

Revision as of 02:16, 13 June 2013

AMPURE RNA beads testing 2, live cells, pure DNA

  • experiment for AMPURE RNA/DNA beads testing worked here, when using normal ratios of 1 to 1 for beads to sample. not sure why. Samples were then used for downstream processing to see sequencing differences when AMPURE RNA beads, DNA beads, and Zymo kit are used for purification. However during multiple instances, purifying with beads resulted in almost complete sample loss, and need to figure out why.


  • in last experiment with AMPURE RNA beads beads testing, live cells there was much sample loss after purification, but recovery was fine with Zymo RNA purification columns. Now trying experiment again and collecting elutions to see where sample loss is. Will also try experiment with different bead to sample ratios since that may be the issue too.


  • if works can use sample for downstream processing. such as the polyA experiment


  • UPDATE for second experiment can bring IVT sample to 50 uL with nuclease free water, then at 2:1 ration of beads (100 uL beads), and follow beads protocol as normal but with short (3 minute) drying time.



Before starting protocols

1. Check if have enough reagents etc for the protocol

  • lysis buffer
  • nextera transposomes
  • transposase/transposome
  • RNA clean and concentrator, AMPURE beads
  • IVT reaction mixture
  • cells etc
  • blocking primer, RNase III, PolyA Polymerase, ATP, taq polymerase, KAPA SYBR FAST, T20VN_PE_R


2. Purify GM12878 DNA from GM12878 cells with DNeasy blood and tissue kit for cells (USE PROTEINASE K (invitrogen) AND protease (qiagen) AND RNAase A). quanititate DNA with nanodrop.

GM12878 DNA purification method used (followed DNeasy kit protocol for washes etc):

a. pellet cells

b. resuspend in:
    1. 100 ul PBS
    2. 10 uL proteinase K
    3. 3 uL 5 AU Qiagen protease
    4. 4 uL A797A promega RNase solution (RNase A)

c. add 200 uL buffer AL, incuabate at 56C for 10 minutes

d. step 3 in DNeasy kit protocol until finish


3. samples this time

Samples (for IVT:
1. 1K cells cells beads T1
2. 1K cells beads T2
3. 6 ng Pure DNA beads T1
4. 6 ng Pure DNA beads T2
5. NTC (no tspn) beads T1
6. NTC (no tspn) beads T2


IVT Protocol

  • If need to make more transposome, do first 2 steps.

1. annealing of ME sequence to T7 transposon sequence

    • a. Make 100 uM stock solution of T7tspn-top2 and T7tspn-bot.
    • b. Incubate 5 uL of each oligo (100uM) with 40 uL EB buffer at 95C for 2 minutes. Oligo's now at 10 uM in 50 uL.
    • c. cool to RT at 0.1 C/s


2. transposome complex generation, run controls!!!

  • add the below components into one tube and incubate for 20 minutes at RT
1.25 uL of annealed transposon
1.25 uL of 100% sterile glycerol
2.50 uL of Ez-TN5 transposase
  • store at -20, is good for a year


3. Prepare samples, lyse cells with lysis buffer


  • Make 10ml 10X lysis buffer (LB, 100mM Tris.Hcl pH 7.5, 100mM NaCl, 30mM MgCl2, 1% NP40, Crawford et al. PNAS 2003) in nuclease free H2O.
  • Prepare aliquots of lymphocytes GM12878 (have GM20431, GM12878, MEFs) that contain 1000, 500, 100 cells.
    • spin down cells to concentrate them as necessary.
  • Prepare 2X LB from 10X buffer. mineral oil optional.
  • if needed make 4 or 6 uL solutions. keep 1:1 ratio of cells:lysis buffer


  • make each cellular concentration in triplicate for AluI amplification with PCR for cell number quantification! store samples and perform quantification later. Use pure DNA nanodropped controls!!
AluI PCR quant
5 uL AluI 245-263 primer
5 uL AluI 21-40 primer
X sample 
X water
_________
25 uL
  • incubate below mixtures at 37C for 30 mins.
Samples (for IVT:
1.  1K cells cells beads T1
2.  1K cells beads T2
3.  6 ng Pure DNA beads T1
4.  6 ng Pure DNA beads T2
5.  NTC (no tspn) beads T1
6.  NTC (no tspn) beads T2


' 1. 1K cells cells beads T1 2. 1K cells beads T2 3. 6 ng Pure DNA beads T1 4. 6 ng Pure DNA beads T2 5. NTC (no tspn) beads T1 6. NTC (no tspn) beads T2
cells 1 ul 1 ul 1ul pure 1 ul pure 1ul N-H2O 1 N-H2O
2X LB 1 ul 1 ul 1 ul 1 ul 1 ul 1 ul


4. transposition reaction, using (T7tspn-top2)

  • add the below into one tube and incubate for 10 minutes at 55C.
1 uL nextera LMW buffer
2 uL lysed/pure genomic DNA (X ng/pg DNA)
1.2 uL Nuclease free water
.8 uL prepared T7 transposomes (MAKE SURE TO ADD LAST) (if was proportional to shendure would use .625 uL)
___________
5 uL total solution
method used in shendure paper:
4 uL nextera HMW buffer
X uL genomic DNA at prepared quantities
X uL Nuclease free water
______
17.5 uL total solution

add 2.5 uL of prepared transposomes


5. Protease digestion of transposase, protease inactivation

To each tube, add:
1 uL  Qiagen Protease, for 5 uL reaction 1 uL of .5 for .1 AU final. (stock is 5 AU and diluted 10X. want .5 AU/uL final [])
Incubate: 50C 10 minutes, 70C 20 minutes


6. Fill in reaction

  • Add 6 uL 2X taq polymerase, run at 72C for 3 minutes. (same as nextera)


7. Maxiscript (Ambion) T7 Protocol, IVT

  • DNA from PCR can be used directly in the MAXIscript Kit without any pretreatment or purification.
a. Thaw 10X Transcription Buffer and ribonucleotide solutions. Store the ribonucleotides (A, C, G, U) 
   on ice, but keep 10X transcription buffer at room temp

b. Assemble reaction mixture at room temperature, ADD IN ORDER AND MIX THOROUGHLY!!!!
  bring to 20 uL with Nuclease free water
  X   uL   DNA template (list 1 ug)
  2   uL   10X Transcription Buffer
  1   uL   10 mM ATP
  1   uL   10 mM CTP
  1   uL   10 mM GTP
  1   uL   10 mM UTP
  2   uL   T7 Enzyme Mix


b. Incubate reactions at 37C overnight for ~16 hours. (>10 uM limiting nucleotide)



8. Clean RNA with with AMPURE RNA beads. elute in 10 uL or less.

  • T1 is normal 1:1 ratio and collecting washes/elutions
  • T2 is add 30 uL N-H20 (50 uL total), then add 2 volumes of beads (100 ul)
  • can quantitate with Qubit or on TBU gel.


Results

  • TBU samples after IVT, and before purification. cell samples have a slightly less smear then usual. Pure DNA samples look normal
' ng/ul intensity '
5 2077.610062
2.5 1089.340032 in 20 uL
1 65.11745272 25842.52077 1302.349054
2 51.66176753 20523.38061 1033.235351
3 165.8200441 65651.06196 3316.400882
4 196.650637 77838.64232 3933.012739




  • For first set of samples, ran AMPURE RNA beads as normal. with 20 uL of IVT reaction, added 20 uL of beads and ran through the procedure.
  • can see that most of the sample is still in the supernatent (flow though), during first step of beads purification. For the pure DNA sample some was eluted in the elution, however most of it is in the flow though.


  • Using first set of trial samples
Samples:
1. 1K cells cells beads T1
3. 6 ng Pure DNA beads T1
5. NTC (no tspn) beads T1
  • TBU on beads washes, elutions etc.
    • 4/40 uL supernatent used
    • 5 uL resuspended beads
    • 1/10 uL elution used

File:ZhangLab 2 2013-06-11 16hr 12min-labeled.jpg



  • For second set of trial samples, first 30 uL of water was added to samples, and then 100 uL of beads for a total of 150 ul in solution. Rest of the beads purification protocol was followed as normal, with ~3 minutes used for drying the beads.
  • here most of the sample is in the elution for both pure DNA and where 1K cells were used. DNA band at ~80 bp is lost, which is what is wanted. The beads still retain some pure DNA sample (lane 5) probably because there is alot of sample. can even try a higher beads ratio.


  • second set of trial samples
Samples:
2. 1K cells beads T2
4. 6 ng Pure DNA beads T2
6. NTC (no tspn) beads T2
  • TBU on beads washes, elutions etc.
    • 4/150 uL supernatent used
    • 5 uL resuspended beads
    • 1/10 uL elution used

File:ZhangLab 2 2013-06-11 19hr 08min-labeled.jpg


  • quantification of results. can see with T1 samples, especially pure DNA sample, most of the sample is in the supernatant. When the altered protocol was used with more beads, most of the sample was in the elution, with ~40-50% sample loss.
' ng/ul intsnity total volume ' ' '
5 2772.840083
2.5 1662.26005 total ng originally in sample loss
1. beads flow through 1. 1K cells T1 9.771560806 4892.520146 38 371.3193106 1302.349054 71.49%
2. beads flow through 3. 6 ng pure DNA T1 105.9414225 47614.25142 38 4025.774055 3316.400882 -21.39%
3. beads flow through 5. NTC (no tspn) T1 -1.067640332 77.40000231 38 0 N/A
4. beads after elution 1. 1K cells T1 2.957868861 1865.660056 5 14.78934431 1302.349054 98.86%
5. beads after elution 3. 6 ng pure DNA T1 9.601221884 4816.850144 5 48.00610942 3316.400882 98.55%
6. beads after elution 5. NTC (no tspn) T1 -1.40309568 -71.62000213 5 0 N/A
7. 1 uL elute 1. 1K cells T1 3.638751823 2168.130065 10 36.38751823 1302.349054 97.21%
8. 1 uL elute 3. 6 ng pure DNA T1 27.48280178 12760.42038 10 274.8280178 3316.400882 91.71%
9. 1 uL elute 5. NTC (no tspn) T1 0.149899152 618.2700184 10 1.498991518 N/A
ng/ul intensity total volume (ul)
5 3113.460093
2.5 2288.280068 total ng originally loss
1. beads flow through 2. 1K cells T2 -3.608182457 272.1400081 150 0 1033.235351 100.00%
2. beads flow through 4. 6 ng pure DNA T2 9.117192613 4472.430133 150 1367.578892 3933.012739 65.23%
3. beads flow through 6. NTC (no tspn) T2 -3.266135873 385.0400115 150 0 N/A
4. beads after elution 2. 1K cells T2 8.192848833 4167.330124 5 40.96424416 1033.235351 96.04%
5. beads after elution 4. 6 ng pure DNA T2 45.27912092 16408.47049 5 226.3956046 3933.012739 94.24%
6. beads after elution 6. NTC (no tspn) T2 -4.005307933 141.0600042 5 0 N/A
7. 1 uL elute 2. 1K cells T2 52.85895199 18910.36056 10 528.5895199 1033.235351 48.84%
8. 1 uL elute 4. 6 ng pure DNA T2 240.3917933 80809.70241 10 2403.917933 3933.012739 38.88%
9. 1 uL elute 6. NTC (no tspn) T2 33.8997552 12652.46038 10 338.997552 N/A


conclusions

  • will now add 30 uL of water was added to samples, and then 100 uL of beads for a total of 150 ul in solution. Rest of the beads purification protocol will be followed as normal, with ~3 minutes used for drying the beads.


  • will process this sample through to test replicates, to see if the polyA issue solved.


  • can do one more trial of beads purification, increasing ratio of beads to sample again to ensure everything is working correctly.