Brandon:LabNotes/Project1/2012-6-15

From ZhangLabWiki
Jump to navigation Jump to search

RNA Fragmentation protocols, Mg++



  • 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 K+ will affect reaction and it is in high concentration.

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

Rnase III 1X buffer

NEB
1X NEBNext RNase III Reaction Buffer: 
10 mM Tris-HCl 
10 mM Mg(Cl)2 
1 mM DTT 
60 mM NaCl 
pH 8.3 @ 25°C


  • 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

1. Retrieve IVT RNA.

  • samples
IVT sample
control RNA
NTC etc


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


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

flaskdjfalkfj
adslfjkalsdf
a
d sf
af
a 
a 
ag


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

  • enzymatics PolyA polymerase.
1. assemble reaction:
   2 uL 5X SMART MMLV first strand buffer (rather than 10X polyA buffer)
   1 uL polyA enzyme
   1 uL 10 mM ATP
   bring to 10 uL with RNA or w/e

2. Incubate at 37C for 10 minutes

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



7. single strand synthesis MMLV RT (Clontech)

20 uL reaction

1. Add 2.5 uL 20 uM primer stock to RNA sample. Bring to final volume of 11.5 uL with
   Nuclease free H2O (one 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.
   4 uL 5X first strand buffer
   2 uL dNTP mix
   2 uL 100 uM DTT
   .5 uL SMART MMLV RT and mix (ADD LAST!!!!!)
  ____
   20 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 or T7-top3-PCR) 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

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

Notes and primers used etc

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) Tm=66.79


To use with (T7-top3) transposon

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