Brandon:LabNotes/Project1/2012-6-15

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RNA Fragmentation protocols, Mg++[edit]



  • fragmentation with Mg++ -> end repair (need 3' end repaired) -> PAP with MMLV buffer -> MMLV RT (with MMLV buffer) -> PCR
  • end repair is the inefficient step. will try a cominbation of phosphatases to repair the 3' ends.

IVT and magnesium compatible so don't have to clean after IVT?

  • Just clean after IVT since not sure how high Tris and NaCl presence will affect magnesium fragmentation and it is in high concentration. (could buffer it)


T7/T3 RNA Polymerase

invitrogen
storage buffer
20 mM Tris-HC1 (pH 7.5)
0.1 M NaCl
0.1 mM EDTA
1 mM DTT
50% (v/v) glycerol
0.01% (w/v) Triton® X-100

reaction buffer
5X T3/T7 Buffer
0.2 M Tris-HCl (pH 8.0)
40 mM MgCl2
10 mM spermidine-(HCl)3
125 mM NaCl

NEB Mg++ fragmentation buffer

NEB
1X NEBNext RNA Fragmentation Reaction Buffer: 
40 mM Tris-OAc
100 mM KOAc
30 mM Mg(OAc)2
pH 8.3 @ 25°C

NEBNext® RNA Fragmentation Stop Solution (10X) (Store at -20°C or 4°C) (#E6187A:   0.4 ml)
1X NEBNext RNA Fragmentation Stop Solution: 
50 mM EDTA


  • Buffer compositions for 3' end repair.

Shrimp Alkaline Phosphatase (SAP) (1 hour at 37C for 3' ends. 65C 15 mins inactivate)

Fermentas
Storage Buffer
25 mM Tris-HCl (pH 7.6 at 4°C)
1 mM MgCl2
0.1 mM ZnCl2 and 50% (v/v) glycerol.

10X Reaction Buffer
100 mM Tris-HCl (pH 7.5 at 37°C)
100 mM MgCl2
1 mg/ml BSA.

Antarctic Phosphatase (37C for 30 mins. inactivate 5 min at 65C)

NEB
Storage Conditions:
10 mM Tris-HCl
1 mM MgCl2
0.01 mM ZnCl2
1 mM Dithiothreitol
50% Glycerol
pH 7.4 @ 25°C

1X Antarctic Phosphatase Reaction Buffer:
50 mM Bis-Tris-Propane-HCl
1 mM MgCl2
0.1 mM ZnCl2
pH 6.0 @ 25°C

T4 Polynucleotide Kinase (PNK) (37C for 30 mins. 65°C for 20 minutes inactivate)

NEB
1X T4 Polynucleotide Kinase Reaction Buffer:
70 mM Tris-HCl
10 mM MgCl2
5 mM Dithiothreitol
pH 7.6 @ 25°C

Storage Conditions:
10 mM Tris-HCl
50 mM KCl
0.1 µM ATP
1 mM Dithiothreitol
0.1 mM EDTA
50% Glycerol
pH 7.4 @ 25°C


  • Shrimp alakaline phosphotase, polynucleotide kinase and antarctic phosphotase buffers
  • magnesium protocol from NEB
  • check if buffers compatible for end repair
  • cleanup before PAP etc?
  • copy paste PAP, MMLV, PCR protocols from what was used with RNase III.


Mg++ protocol[edit]

1. Retrieve IVT RNA.

  • samples
1. Antarctic, SAP and PNK treated
2. Antarctic, SAP and PNK treated (meant to only do antarctic but forgot)
3. NTC
4. clontech control RNA

used 2 uL each for samples 1, 2. Used RNA from #10 (top2)


2. Perform EtOH cleanup of RNA. (or do columns?)


3. Perform magnesium fragmentation (NEB)

a. set up fragmentation buffer

 1-9 uL Purified RNA
 1   uL RNA fragmentation buffer (10X)
 X   Nuclease free H2O

b. Incubate in a preheated thermal cycler for 1-5 minutes at 94C.

c. Transfer tube to ice

d. Add 1 uL 10X RNA fragmentation stop solution 


4. EtOH cleanup or RNA column purification

  • resuspend in 8 uL


5. Perform End repair of 3' End. (SAP, PNK, Antarctic Phosphotase)

a. Add antarctic phosphotase
   1 uL of 10X buffer
   1 uL of 5U/ul antarctic phosphotase

b. Incubate at 37C for 30 minutes
 
c. mix SAP and PNK buffters together, before addition
   .5 uL of mixed buffer
   1  uL SAP
   1  uL PNK
   2.5 uL nucleaes free H2O
   
d. Incubate at 37C for 1 hour. (SAP needs 1 hour for 3' end phosphate removal)

e. Incubate at 65C for 20 minutes for inactivation.


6. Poly(A) Addition with polyA polymerase (Enzymatics)

  • enzymatics PolyA polymerase.


a. Add 2 uL of 100 uM DTT (with MMLV reagents)

b. assemble reaction:
   2   uL 5X SMART MMLV first strand buffer (rather than 10X polyA buffer)
   1.5 uL polyA enzyme
   1.5 uL 10 mM ATP
   (21 uL reaction now, in end will have ~50 mM Tris, 40 mM Kcl, 6 mM MgCl2, ~11 mM DTT, compatible with MMLV)

c. Incubate at 37C for 10 minutes

d. Heat inactivate at 70C for 20 minutes. (rui and NEB)



7. single strand synthesis MMLV RT (Clontech)

20 uL reaction

1. Add 5 uL 20 uM primer stock to RNA sample. Final Volume = 25 uL (primer from BENG160 class, T20VN_PE_R)

2. Heat the mixture to 70C fo 3 minutes. Immediately cool on ice.

3. Add the following to the reaction.
   2 uL 5X first strand buffer
   3 uL dNTP mix
   1 uL 100 uM DTT (keep at final [] at 10 uM)
   .75 uL SMART MMLV RT and mix (ADD LAST!!!!!)
  ____
   32 uL total

4. Incuvate at 42C for 60 minutes

5. Terminate the reaction by heating at 70C for 10 minutes



8. second strand synthesis (qPCR) (KAPA)

KAPA SYBR FAST qPCR mix X35 cycles

25 uL KAPA SYBR
4    uL primers, 1 uL F, 1 uL R (T7-top2-PCR-iaf or T7-top3-PCR-iaf) and (PCR_R.N2Ind[XX] (23,24))
1  uL H2O
20    uL DNA template (use whole RT reaction)

KAPA SYBR cycles:
98C 3min, (98C for 30s, 60C for 30s, 72C for 1 min) X35, 72C for 5 min, 4C forever

  • terminate when curves saturate


9.qiaquick cleanup OR beads purification (this time ran beads purification)

  • run on gel or w/e
  • run qiaquick to clean sample before performing qPCR.
    • 6 enzymes from 6 different reactions in there already.


results[edit]

  • performed RT with T20VN_PE_R, and PCR with (T7-top2-PCR-iaf, PCR_R.N2Ind[24]
  • nanodrop quant:
Mg++ ng/ul
9 67.11
10 61.81
11 10.53
12 11.56
  • nonamer at 42C has index22
  • nonamer at 16C then 42C has index23
  • Mg++ fragmentation shows a quality smear for both samples, which were successfully PCR amplified. according to the gel smears


qPCr curves upon amplification

  • Mg++ samples showed weird curves. NTCs are correct, though could be something wrong with the SYBR green reading etc. nanodrop values after beads purification are fine, and gel smear is fine.
  • samples 9,10,11,12 are the Mg++ fragmentation samples.

File:2012-06-22 qPCR after nonamer1, nonamer2 and mg++ run with plate-labeled.bmp

  • TBE gel

File:ZhangLab 2 2012-06-22 17hr 25min-labeled.jpg


  • TBU gel of same samples shows smears still present with nonamers, but better fragmentation sizes for the Mg++ fragmentation

File:ZhangLab 2 2012-06-22 18hr 41min-labeled.jpg


  • proceeding to cloning and transformation to see if properly labeled fragments are obtained that contain genomic DNA.

More REsults[edit]

Notes and primers used etc[edit]

Stuff used for RNA-seq for BENG160 class

5’ end addition primers

TSO_N10_BC[XX]
[AAGCAGTGGTATCAACGCAGAGdUdU]NNNNNNNNNNTTTAGGrGrGrG
         adatpor1

5'- AAG CAG TGG TAT CAA CGC AGA G/ideoxyU//ideoxyU/ NNN NNN NNN NTT TAG GrGrGrG -3'

P1-STRT
5’- [AATGATACGGCGACCACCGA][GATCT][AAGCAGTGGTATCAACGCAGAGT] -3’  (Tm=82.59)
      ILA adaptor blue Tm=64.31         adaptor1 Tm=61.02

STRT-SEQ
5'- [GATCT][AAGCAGTGGTATCAACGCAGAGTT] -3'
                adaptor1
3’ end addition primers

T20VN_PE_R
5'-Bio-[GCATTCCTGCTGAACCGCTCTT]CCGATCTTTTTTTTTTTTTTTTTTTTTVN  -3’  (Tm=78.92)
          adaptor2 Tm=65.68

PCR_R.N2Ind[XX] (XX=23,24 for me)
5’- [CAAGCAGAAGACGGCATACGAGAT][TACAAG]CTCG][GCATTCCTGCTGAACCGCTCTT] -3’ (Tm=87.12)
        ILA adaptor orange       bc          adaptor2 Tm=65.68
My modifications for T7-tspns for 5' addition primers
 
To use with (T7-top and T7-top2) transposons

5’- [AATGATACGGCGACCACCGA][GATCT][CTCCCTCGCGCCATCAGAGAT]  -3’  (Tm=86.59)
     ILA adaptor blue           Top2-5’end (T7-top2-PCR-iaf) Tm=66.79
                               (can be used if top1 is used for transposition)


To use with (T7-top3) transposon

5’- [AATGATACGGCGACCACCGA][GATCT][GGGAGACATTAAGATGTGTATAAGAGACAG] -3’  (Tm=81.40)
     ILA adaptor blue              Top3-5’end (T7-top3-PCR-iaf) Tm=60.71