Brandon:LabNotes/Project1/2013-6-10
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AMPURE 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 |
- 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
- second set of trial samples
Samples: 2. 1K cells beads T2 4. 6 ng Pure DNA beads T2 6. NTC (no tspn) beads T2