Lab Techniques (Research)

FYI: I m attached to research lab under the Cellular and Molecular Research division. My lab focuses on gene structure and expression.

I was assigned to do a project to prevent 2 proteins from interacting to prevent tumorigenesis.
Throughout my SIP, I did different things everyday unless I don’t get any expected results and need to redo. My work rotates around Mbio, CellBio, MCT and Biochemistry. I am going to focus on DNA preparation in my discussion here. I am supposed to isolate the DNA I clone from the host cells that I had transformed.

There are three types of DNA prep for plasmid isolation: maxi-, mini, midi-prep.

Below are differences between these 3 prep works:

As I only did the maxi- and miniprep, I cannot comment more on midiprep.

Maxiprep:
-large scale DNA preparation
-Quantity of DNA eluted: 500ug/ml- 850ug/ml
Steps and reagents involved:
-Uses cultured cell clones in 250ml of LB media
- Requires centrifugation at 4۫C
- Requires a big Maxi- column to bind DNA, a dish to collect flow through that pass the column for disposal and a Du-pont tube for DNA elution
- Steps are lengthy and more buffers are involved like W8 (wash buffer), E4 (elution buffer), 70% ethanol for precipitation, TE buffer for resuspension and Equilibration Buffer (EQ1) to wet the column before draining any solution
- Drain through column by gravity flow
- Principle is lysis, bind and wash DNA, precipitation and elution.Details of the protocol can be found in www.invitrogen.com under PureLink™ HiPure Plasmid Filter Purification
Miniprep
-Small scale DNA preparation
-Quantity of DNA: up to 40ug/ml
-Steps and reagent involved:
-Uses cultured cells in 4ml of LB media
-Only requires a normal centrifuge
- Only requires a normal spin column to bind DNA and 2ml Wash tube to elute the DNA and for flow through collection
-Involves lesser steps and buffer and column work by centrifugation to bring the solution down.
- Principle works by alkaline lysis of the host cells, bind, wash and elution purified DNA.
- Details of the protocol can be found in www.invitrogen.com under PureLink™ Quick Plasmid Miniprep Kit
Midiprep
-Middle scale DNA preparation
-Quantity of DNA: 100-350ug/ml

After the prep for our DNA purification, I need to do a quantitation of DNA content using a spectrophotometer to check is there sufficient DNA for the following steps of the project. Quality of DNA is also monitored. DNA with ratio measured at 1.6-2.0 are considered pure and good quality. Sometimes, ratio may not be quantitated by the spectrophotometer if the concentration is too low. The Miniprep is done before maxi prep so that we can prepare our large amount of DNA with the correct sequence based on the sequence results of the clone from miniprep. We quantitate the DNA in miniprep for sequencing reaction to monitor if the DNA cloned has the right sequence so that we can proceed on our project. 1 ug/ml is sufficient.
For maxiprep, we check the concentration of DNA to see if there are enough for in vitro translation for translating DNA to proteins in the next part of project .
Running a gel with the same aliquot of DNA can confirm the quality of DNA based on comparison of the intensity of the bands to make sure the measurement from the spectrophotometer is accurate. DNA might be contaminated with RNA, proteins and carbohydrates which can be determined by the quality ratio of the DNA and confirmed again using the gel.

I hope u all understand what I said and dont hesitate to ask. Thanks.

Ai Tee
0503160D
TG01