Sunday, October 21, 2007

Lab Techniques

Hi guys...today i'll be talking about the washing procedures that Zahirah and I always have to do when there is insufficient sequencing plates and Axymats for our experiments. Hee... Feel free to ask any questions ya...

Title: Cleaning of sequencing plates procedure


  1. Flood labeling on the sequencing plates with DMSO and wiping the sequencing plates dry and clean with tissue papers.
  2. Rinse sequencing plates with tap H2O.
  3. Add decon 90into sonicator.
  4. Place all sequencing plates into sonicator. Ensure they are totally submerged by using Styrofoam to press them down and the close the lid of the sonicator. Then add distilled H20 into the sonicator, filling it ¾ full.
  5. Turn on the sonicator for 30min. (It will induce electrical impulses for washing of sequencing plates.)
  6. After washing for 30min, turn on the tap of sonicator to allow the washing solution to be drained out into an empty pail and later been disposed into the sink.
  7. Add decon 90 into sonicator again.
  8. Rinse sequencing plates with tap H20 again.
  9. Place sequencing plates into sonicator and add distilled H20,filling it ¾ full again. Place Styrofoam to press down the sequencing plates to ensure that they are fully submerged into the washing solution.
  10. Turn on sonicator for 15 min.
  11. After 15 min, turn on the tap of the sonicator to drain off the washing solution.
  12. Rinse sequencing plates with tap H2O again.
  13. Take a beaker to fill up with milliQ H2O.
  14. Rinse the sequencing plates in milliQ H2O for 4-5times.
  15. Dry them using tissue papers.
  16. Place them in the oven(65oC) for 1 hour.
  17. Wrap them with aluminium foil and sent them for autoclave.

Title: Cleaning of Axymats procedure

  1. Prepare 0.5% to 1% sodium hypochlorite (bleach) solution. In this case, 1% bleach is prepared using M1V1=M2V2.

5.25% of bleach (stock conc) x V1= 1% of bleach x 1500ml (total volume of bleach solution required)

Thus, V1= 286ml (approx. 300ml)

Hence, add 300ml of 5.25% bleach into 1200ml of milliQ H20 and pour the solution into the sonicator. Turn on sonicator for 15 min after placing the Axymats into the prepared 1% bleach solution.

  1. Rinse Axymats into 70% isopropanol and allow them to dry on tissue paper.
  2. Place them in the oven for 30 min.
  3. Wrap them with aluminium foil and sent for autoclave.



Michelle (0503808h)
TG02

11 comments:

Debra said...

Hi!

Just would like to know what is the purpose of DMSO in this case. Also, i understand it is a MDSD chemical, so do you work in fume hoods for this?

Also, doesn't DMSO increase the chances of DNA mutation? I'd imagine it'll be a pain when it comes to sequencing reactions if not removed properly. If so, is there any other alternatives that other labs may perhaps use, or is DMSO univeral in this case?

Thanks!

- Debra, TG02

The Lab Freaks said...

hi...

maybe i know why is it that u place them in an oven before autoclaving it? what is the purpose of placing them in an oven first?

~Jeremy~
TG01

Vino said...

hey hi

Hey i dun really quite understand abt the Axymats.. wat issit for? dun mind canu elaborate more on it?

Vino
TGO2

royal physicians said...

Hi michelle,

I would ask how does the electrical impulses produce by the sonicator aid in washing the plates??

Avery
TG02

VASTYJ said...
This comment has been removed by the author.
VASTYJ said...
This comment has been removed by the author.
VASTYJ said...
This comment has been removed by the author.
VASTYJ said...

Hi Michelle,

Your post is quite difficult to understand without pictorial presentations. =) hehe. Because I'm rather uncertain of what is going on, I shall ask you two fundamental questions in respect to your post: What are Axymats and sequencing plates?

Loh Sharon, Tg 01

first6weeks said...

Hello!

May i know what's the purpose of rinsing Axymats in 70% isopropanol? And what are Axymats? Thanks!

June, TG02

J.A.M.M.Y.S said...

Hiya pple,

Firstly, Axymats are silicon mats that are used to cover the sequencing plates. The Axymats prevent the evaporation of the contents in the wells of the sequencing plates,while sequencing plates are the 96 wells plates used for DNA sequencing.

We only used the DMSO to remove the labelings that we have wrote using marker and according to our lab officer,we do not need to perform this step in the fume hood. DMSO is used because it is a stronger solvent than ethanol and the labelings cannot be removed using ethanol.

We placed the sequencing plates into the sonicator because the electrical impulses produced will dislodge the DNA pellets and other remianing debris from the wells of the plates thus this ensure that the plates are clean.

We soaked the Axymats in 70% isopropanol because we want to remove the DNA that have been evaporated during the experiment and stuck on the mats by denaturing it.We used 70% isopropanol instead of 100% because if 100% isopropanol is used,it will be easily evaporated off and won't have much effect in removing them.

Lastly, we placed the wash Axymats and the sequencing plates in the oven so that they can be dry before wrapping them in aluminium foil and sending them for autoclave.


I hope I'm able to clarify yr doubts ya...

Michelle
0503808H
TG02

royal physicians said...

Hey Michelle,

Just wanna ask. someone mentioned that DMSO could be a factor that increases DNA mutation right? SO does it actually affect the sequencing after that? ALso, you mentioned that sonication is used to remove unwanted debris and such so does it remove any remaining DMSO on the plates?

Thanx,
Johanna
0503309G