Molecular Biology

Making miRNA sensors

Create MSCV-PGK-hCD25 GFP (or CFP) miRNA sensors

Genotyping - Ear Punch DNA

Genotyping  - Ear Punch DNA  (Ansel Lab 2015)


Mouse Tech 


1. Place ear punches directly into pre-labeled PCR strips, always left-to-right.

            Don’t combine > 1 cage per strip (leave empty wells if

2. Spin briefly in tabletop centrifuge to pellet tissue (Laura’s bench)

3. Add 20 ul ear punch digestion buffer w/proteinase K per well.

            20 ul reaction = 19 ul ear digestion buffer + 1 ul proteinase K

Ear Punch Processing for PCR

Collecting Ear punches:

1.  Mice can be anesthetized or non-anesthetized for punching.  If you have to do a lot, then anesthesia may help the processes move more quickly.

2.  Ear punch material can be collected into eppendorf tubes for DNA extraction using forceps.  Clean instruments and area with dilute bleach between each punch to prevent cross-contamination.

3.  Store ear punches at -20oC or process immediately.  DNA degradation may occur if the specimens are left at RT.

Polysome analysis by sucrose gradient fractionation

This protocol describes how to carry out sucrose gradient fractionation of NK cell lysate to look at polysome prolifes


A. Magnetic beads- antibody preparation (Day 1)

1. Take 50ul or 10ul of beads/ ChIP condition 

Note- volumes vary depending on the size of the ChIP. We take 50ul of beads for ~7 million cells chromatin for NFAT/POL2 ChIPs, and only 10ul of beads for ~1 million cell chromatin for H3K4me2.

DNA fragmentation by sonication (for ChIP)

1. Fill up bioruptor tank with ice.

2. Take samples out of freezer and keep them on dry ice- they must not thaw!

3. Prepare fresh a stock of complete lysis buffer: (N+1)x120ul lysis buffer +NaBu (20ul/ml) + PI (10ul/ml) + PMSF (10ul/ml)- keep at RT.

4. Add 120ul of complete lysis buffer to each sample- allow the pellet to thaw at room temperature into the buffer (1-2 min). Resuspend the pellet until the solution is clear- avoid forming bubbles.

5. Incubate on ice for 5 minutes.

Cell fixation for ChIP

1. Work at room temp- make cell suspension at concentration of 1-2x106 cells/ml of complete medium (with FCS) in a falcon 15ml if 10mls.

2. Vortex cell suspension at medium speed and add drop by drop 1:10 of 10x fixation buffer.