Rna Probe Preparation

Apr 22, 2021

Making an RNA probe for in situ (by Ehab Lab)

Template preparation.

Method 1: Do a PCR with M13F and M13R on the plasmid, Check that the insert has been amplified and purify using PCR purification kit Use as template in next reaction

Method 2: Check for orientation of your gene using sequence analysis Determine which RNA polymerase will be used SP6 or T7 (This is done by checking the orientation of the gene 5’ end is where ATG start codon is and 3’ end is where the stop codon is.) Digest with appropriate enzyme to cut the plasmid for 2 hours (e.g., SpeI or Sac-I or NdeI or Sal-I digest is compatible with T7 polymerase Sph-I, or Nco-I or Sac-II digest is compatible with SP6 polymerase)

Purify the digested plasmid using PCR purification kit Use in the next reaction

Probe Preparation

  1. Prepare the following mix:
Reagents Amounts
10X Transcription Buffer 2uL
RNAse Out 1uL
DIG-RNA labeling mix 2uL
Template DNA ~500ng
RNA Polymerase 2uL
H2O up to 20uL
  1. Incubate 2-4 hours at 37°C
  2. Add 1μl DNase (RNase free) and incubate 10 min at 37°C
  3. Check 1μl on a gel
  4. Purify probe immediately (with RNA purification kit or phenol:chloroform:isoamylalcohol)
  5. Check 1μl on a gel
  6. Add 30μl of Hybridization solution on the probe and store at -80°C