TEAM ALBERTA

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Beads

Bead Preparation and DNA Isolation Protocol Modifications

• May 25, 2010:

We will purchase NEB oligo dT iron micro beads and cellulose beads, which have a polyT tail (5' to 3') 25 nucleotides long. They are meant for mRNA isolation, but should work fine for our purposes.

• May 27, 2010:

No binding capacity specified in the manual for the beads in terms of mols or mass of DNA to beads. Binding capacity is given in terms of cells because the beads are meant for mRNA isolation from cells. (We will have to quantify binding capacity later).

Derivation of mole matching equation:

ssDNA is ~330 grams/(mol*bp) moles = [concentration X volume] / [(330grams/mol X bp) X length]

moles1 = moles2 c1v1/[(330g/mol X bp) X l1] = c2v2/[(330g/mol X bp) X l2]

v1 = v2(c2 X l1/ c1 X l2)

• June 8, 2010:

Produce a modified protocol for Anchor binding to cellulose beads.

Protocol Preparation:

Allow everything to come to room temperature.

Spin 2000 to 5000g of beads in microcentrifuge for 10 seconds.

Remove supernatant without disturbing the beads.

Add 200μl of Wash Buffer to beads and agitate.

Centrifuge for 10 seconds and remove supernatant.

Prewarm Elution Buffer in a 70^oC water bath.

Isolation Procedure:

Apply DNA Anchor to cellulose beads, agitate and let it stand at room temperature for 5 minutes.

Microcentrifuge for 10 seconds.

Pipette supernatant back into original microcentrifuge.

[Add 4μl Wash Buffer to beads and agitate to resuspend.

Transfer beads and wash buffer to column reservoir of spin column.

Let it stand at room temperature for 2 minutes while agitating. Microcentrifuge for 10 seconds.] X3  ::Add 400μl Low Salt Buffer, resuspend by agitation for 2 seconds and microcentrifuge for 10 seconds.  ::Transfer and place spin column reservoir in a clean microcentrifuge tube.

[Add 200μl pre-warmed Elution :Buffer to column reservoir. Agitate to resuspend beads and let it stand for 2 minutes. Microcentrifuge for 10 seconds] X2

Place eluent on ice.

• June 9, 2010:

Calculated the mass of beads to determine the range of mg RNA could be isolated.

0.06g beads = 0.0599g; therefore, 0.06g can isolated 0.1 to 1 mg RNA.

Calculated the minimal and maximal rpm for 2000 to 5000g of beads using the following formula:

a = 4(pi)2r(rpm)2 / 602

So, when centrifuging the beads, the rpm must stay between 4700 to 7400 rpm.

In the modified mRNA procedure, the centrifuge is set to 5500 rpm.

Binding Capacity Testing for Cellulose Beads

• June 10, 2010:

Attempted to find binding capacity (in moles per mass of beads) by binding known masses of anchor to the bead, then quantifying the mass of DNA recovered after eluting it off of the bead. Measuring precise volumes of the beads was done by pipetting the cellulose bead slush into PCR tubes that were marked at 16 uL. We quantified the masses of DNA using the nanodrop 1000 spectrophotometer that:

(1) did not bind to the bead,

(2) came through the washes, and (3) were eluted from the beads.

Results: Most of the anchor did not bind to the cellulose beads, and a very small amount of DNA was bound and eluted off of the beads. Possible reasons could be that the anchor was not binding, or there was too much anchor relative to the beads. Also, there were issues with the cellulose beads settling very quickly without agitation. Gel electrophoresis showed that there was a lot of anchor that did not bind, and no anchor band was visible in the elution lane (which was expected based on the nanodrop reading).

• June 11, 2010:

Re-attempt to find the binding capacity using 1500 ng of anchor DNA to 16 uL of cellulose bead slush.

Results: There were no significant nanodrop readings, except for the measurement of the amount of DNA that did not bind to the beads. Based on the nanodrop reading, over 1100 ng of DNA did not bind to the beads. The rest must have been washed away in the wash. Perhaps the melting temperature of the polyA12 tail is too low.

• June 14, 2010:

Attempted to bind 500, 1000, and 1500 ng of DNA to anchor bound to 16 uL of beads.

Results: Based on nanodrop readings, we recovered 560 ng from 1500 ng of initial DNA, 63 ng of 1000 ng of initial DNA, and 244 ng of 500 ng of DNA. There were strange results of the 1000 ng of DNA, but a good sign that something appears to be binding to the cellulose beads.

To attempt to find the amount of anchor that will saturate the beads binding capacity, we used 1, 1.5, 2, and 2.5 ug of anchor and attempted to bind them to 16 uL of cellulose beads.

Results: Nanodrop readings multiplied by volume gave 600, 763, 1102, and 1730 ng of DNA not binding to the bead. What bound to the bead was 248, 1935, 4032, and 2470 ng of DNA. Obviously, something has caused the DNA readings to imply that more DNA was recovered than was put in initially. Later on, we found out that the readings are extremely sensitive to the small amount of residual washing solutions left over, causing the blanking process of the nanodrop to not accurately represent what solution the DNA was in.

Binding Capacity Testing for Iron Micro Beads

• June 15, 2010:

Attempt to bind 100, 500, 1000, and 2500 ng of polyA12 anchor to 20 uL of polyT iron micro beads.

Results: 80, 250, 305, and 460 ng of DNA were recovered from the beads after applying 100, 500, 1000 and 2500 ng of DNA initially. The iron micro beads appear to be more efficient at lower masses of DNA being bound. This appears encouraging; however, the nanodrop readings multiplied by the solution volume for the amount of DNA, which did not bind initially were 335, 785, 970, and 2140 ng of DNA. The amount of DNA not bound and the amount that did bind does not add up to the amount of DNA added. This could be due to the residual solution left behind after washes. We will redo the experiment with less concentrated anchor and careful washes.

• June 16, 2010:

Attempt to bind 500, 1000, 2500, and 5000 ng of anchor to 20 uL of beads.

Results: From nanodrop readings, the mass of the DNA which didn't bind exceeded the mass of what was initially mixed with the beads. This is obviously an unknown error in measurement . The amount of DNA recovered was 0, 90, 70, and 250 ng of DNA. Looks like nothing bound to the beads. Possible cause of no DNA binding to bead: very low concentration of anchor meant a large reaction volume which would lead to a higher reaction time. Next time, use higher concentration anchor and increase binding time.

• June 17, 2010:

Tested Absorbance of Water with Iron Micro Beads to determine what Buffer solution skewing the nanodrop readings.

Results: Traces of Lysis Buffer skews readings on Nanodrop. For next experiment, use Washing Buffer instead of Lysis Buffer.

Continue testing the binding capacity by using approximately 250, 500, 1000, 2000 ng of anchor to 20μL of beads. :Nanodrop samples during different stages of the modified procedure.

Results: Saturation point was reached before 486 ng for 100μL beads. Next experiment, test higher volumes of anchor with more volume of beads.