The goal of this procedure is to ease retrieval of samples in cryo boxes and cryo vials from duo tanks filled with liquid nitrogen using an easy to construct device. This is accomplished by first removing one or two freezer racks from the doer to allow room for maneuvering and inserting the device vertically into the doer. The second step is to gently scrape along the bottom of the doer with the device.
Next, pull the device upright along the walls of the doer To scoop the freezer box outta the tank, the final step is to grab the box retrieved from the bottom of the doer and replace the removed freezer racks. The main advantage of this technique over existing methods such as decanting, the bar full of liquid nitrogen is that it provides an easy, safe, and rapid alternative, which preserves the sample integrity and can be carried out by a single operator. To begin assembly of the cryo tolerant device, use pliers to straighten out one side of a three-sided strong tie to make an LHA strong tie.
As seen here, the dimensions of the strong tie can be selected depending on the diameter of the neck of the doer. Two such strong ties should be made to allow the device to retrieve five and a half by five and a half inch cryo boxes. Secure these with a strong tie TST strap using a crown bolt, nut washer, and screw to form the base of the cryo scoop.
The device should now appear as seen here. Now using a slotted plate, align the slots on the strong tie tee strapp with the slots on one end of the slotted plate. Then secure the two together with Hillman nuts and bolts.
Drill holes for attachment if the slotted plate and strong ties do not have pre-drilled holes. If desired, a handle can be formed by bending the other end of the slotted plate and wrapping with foam. To better grasp the device, be sure to remove the foam prior to water cla.
The cryo tolerant DIY retrieval device can also be adapted for retrieval of cryo vials. This involves substituting the flat base with a stainless steel strainer altered for scooping vials out from liquid. To begin assembly, first unscrew the crown nuts and bolts, attaching the strong tire tee strapp to the slotted plate.
Then before attaching the stainless steel strainer to the slotted plate, gently bent the neck of the strainer with the help of pliers to orient the base at an angle to resemble a ladle. If the strainer used comes with a lip use pliers to bend the lip down. This will make maneuvering with the device easier.
Align the hole on the handle of the strainer along with the holes on the slotted plate and secure the strainer using Hillman nuts and bolts. Drill holes if the strainer does not come with pre-drilled holes. To begin retrieving cryo boxes from a doer, be sure to first put on cryogenic gloves.
Then remove one or two freezer racks from the doer to allow room for maneuvering with the device. Next, use the upright cryo tolerant DIY retrieval device fitted with the strong tie ttra base to gently scrape along the bottom of the doer and collect the box, fall into the bottom of the doer tank. Next, pull the device upright along the walls of the doer to scoop the freezer box outta the tank.
Note that the device should be held vertically and pulled straight up to avoid the box from slipping back into the doer. To retrieve cryo vials, use the cryo scoop fitted with a strainer to gently scrape the bottom of the doer similar to cryo box retrieval, pull the device upright and grab the vials collected in the strainer. A major challenge in gene expression studies is that the quality of RNA depend on the storage conditions for both frozen tissue samples and extracted RNA.
This figure shows the effect of sample handling on integrity of total RNA total RNA from the same tissue source was subjected to different sample handling prior to the experiment intact total, RNA prepar rate shows clear 28 s and 18 s ribosomal, RNA bands with the 28 s ribosomal RNA band, approximately twice as intense as the 18 s ribosomal RNA band. This is an indication that the RNA is intact. Lanes three and four depict samples with significant degradation due to repeated partial de freezing by short-term retrievals from the doer, thus modeling long-term storage with periodic decanting due to necessity of the retrieval of the fallen vials on boxes.
However, samples stored in the doer service using the DIY device and thus no partial de freezing contain intact RNA in lane two. After watching this video, you should have a good understanding of how to retrieve samples from the liquid nitrogen filled dvar without the need to decant it.
