User:Pranav Rathi/Notebook/OT/2011/01/10/DNA tethering experiments

Attempt .1 (jan/10/2011)

I attempt tethering again today with new beads and new anti-dig.

Ant-dig

Dr. Koch and I prepared new anti-dig: 1X concentration

PBS [1]180μl + aliquots 20μl = [MIX] = Anti-dig 200μl.

PBS was used from the glass door fridge and aliquot from super cold gray fridge. I used two different beads:

1 μm new beads (looks good in microscope), 20μl from the batch. 5μl (batch) +4 5μl BGB = 50μl.
.5μm (FL) expired on 01/07/2010.5μl (batch)+45μl BGB = 50μl.

Procedure[2]

Flow anti-dig 10μl wait for 5 min.
Flow BGB 50μl wait for 2 min.
Flow 1.1kb (new)10μl wait for 14 min.
Flow BGB wait none.
Flow micro-spheres one in each chamber 10μl wait for 14 min.
Flow BGB 50μl + seal it with nail polish

Results

1μM BEAD.[3]

Attempt .2 (jan/11/2011)

components

.5μm bead with 1.1kb dna (ant just made), duplex.

5μl .5 μm bead + 45μl BGB =50μl (1:10).
1.1kb dna (1:100) and (1.5:100).

Procedure[4]

Flow anti-dig 10μl wait for 5 min.
Flow BGB 50μl wait for 2 min.
Flow 1.1kb (new)10μl wait for 14 min.
Flow BGB wait none.
Flow micro-spheres one in each chamber 10μl wait for 14 min.
Flow BGB 50μl + seal it with nail polish

Results

Both the chambers were same no difference between 1 and 1.5 to 100 concentration. Hardly any tethers. The one found, break immediately.

Attempt .3 (jan/12/2011)

components

.5μm bead with 1.1kb dna (ant just made), duplex.

5μl .5 μm bead + 45μl BGB =50μl (1:10).
1.1kb dna (1:100) and (1.5:100).

Procedure

Same as above.

Results

There were some tethers but no stretchable.

Attempt .5 (jan/13/2011)

components

.5μm bead with 1.1kb dna (ant just made), duplex.

5μl .5 μm bead + 45μl BGB =50μl (1:10).
1.1kb dna (1:100) and (2:100).

Procedure

Same as above.

Results

Tethers but no stretching.

Attempt .5 (jan/19/2011)

components

.5μm bead with 1.1kb dna (ant just made), duplex.

5μl .5 μm bead + 45μl BGB =50μl (1:10).
1.1kb dna (1:100) and (5:100).

Procedure

Same as above.

Results

Some Tethers but no stretching.

Attempt .6 (jan/20/2011)

components

.5μm bead with 1.1kb dna (ant just made), duplex.

5μl .5 μm bead + 45μl BGB =50μl (1:10).
1.1kb dna (1:100) and (5:100).

Procedure

Same as above.

Results

Some Tethers but no stretching.

Attempt .7 (jan/24/2011)

components

.5μm bead with 1.1kb dna (ant just made), duplex.

5μl .5 μm bead + 45μl BGB =50μl (1:10).
1.1kb dna (1:100) and (5:100).

Procedure

Same as above.

Results

Some Tethers but no significant stretching. It looked like some beads had something but nothing significant.

Attempt .8 (jan/26/2011)

components

.5μm bead with 1.1kb dna (ant just made), duplex.

5μl .5 μm bead + 45μl BGB =50μl (1:10).
1.5 μl dna + 98.5 μl POP = 100μl dna. 1.1kb dna (1:100) and (1.5:100).

Procedure

Same as above.

Results

I got possible stretching but for sure no over stretching, as bead broke off every time. [5]

Attempt .9 (jan/27/2011)

components

Made new anti-dig (after 14 days of previous one):

180μl PBS + 20μl (anti-dig) = 200μl anti-dig. .5μm bead with 1.1kb dna (ant just made), duplex.

5μl .5 μm bead + 45μl BGB =50μl (1:10).
1.5 μl dna + 98.5 μl POP = 100μl dna. 1.1kb dna (1:100) and (1.5:100).

Procedure

Same as above.

Results

same result as the above.

Attempt .10(jan/28/2011)

components

1μm bead with 1.1kb dna (ant just made), duplex.

1μl .5 μm bead + 45μl BGB =50μl (1:10).
1 μl dna + 98.5 μl POP = 100μl dna. 1.1kb dna (1:100) and (1.5:100).

Procedure

Same as above.

Results

Tried fine tether center to work. The settings are: [6].

Attempt .11(feb/03/2011)

It looks like that i had some tethers in previous samples but could not stretch may be because of not enough stiffness with .5μm beads. So i am trying tethering with 1μm beads. Since big beads are hard to tether because of mutual repulsion with surface i am trying them with different buffer settings (different NaCl concentration). I prepared POP with:

2X ===> 296.5 μl pop + 3.5 μl NaCl (4molar stock) = 300 ul POP buffer.
4X ===> 288.8 μl pop + 11.25 μl NaCl (4molar stock) = 300 ul POP buffer.
6X ===> 281.25 μl pop + 18.75 μl NaCl (4molar stock) = 300 ul POP buffer.
10X ===> 266.25 μl pop + 33.75 μl NaCl (4molar stock) = 300 ul POP buffer.

components

1μm bead with 1.1kb dna (ant just made), duplex.

1μl bead with:

19 μl 2X pop + 1 ul bead (washed stock)= 20μl
19 μl 4X pop + 1 ul bead (washed stock)= 20μl
19 μl 6X pop + 1 ul bead (washed stock)= 20μl
19 μl 10X pop + 1 ul bead (washed stock)= 20μl

1.1kb dna with :

9.9μl 2X pop + .1μl dna = 10 μl (1:100)
9.9μl 4X pop + .1μl dna = 10 μl (1:100)

Procedure

NOTE: keep the x concentration same over the micro-sphere, dna and flow solution in each chamber)

Flow anti-dig 10μl wait for 5 min in both chambers .
Flow BGB 50μl wait for 2 min in both chambers .
Flow 1.1kb (new with 2X or same X concentration POP)one in one and (4X OR other concentration) in second chamber; 10μl wait for 14 min.
Flow POP 50 μl( 2X or same X concentration POP)one in one and (4X OR other concentration) in second chamber; wait none.
Flow micro-spheres (with 2X or same X concentration POP) one in one and (4X OR other concentration) in second chamber; 10μl wait for 14 min.
Flow POP 50μl (2X or same X concentration POP)one in one and (4X OR other concentration) in second chamber + seal it with nail polish

Results

The effort of bringing big beads close to the surface was successful i tried 2x and 4x. In 4x there were more stuck beads than 2x, which shows that higher concentration of salt can help in bringing beads close to the surface, BUT there were no tethers. So i will try different concentrations lets see...

Attempt .12(feb/04/2011)

Everything is same as attempt 11.

Components

9μl 2X pop + 1μl dna = 10 μl (1:10)
9μl 4X pop + 1μl dna = 10 μl (1:10)

Procedure

Similar to 11.

results

I tried 10 times the dna concentration in hope of tethering. But no success.

Attempt .13(feb/07/2011)

I used Ant's old dna (07/15/09) of 1.1 and 4.4 kb.

Components

1μl .5 μm bead + 45μl BGB =50μl (1:100).
.3 μl dna (1.1kb) + 29.7 μl POP(1X) = 30μl dna. 1.1kb dna (1:100).
.3 μl dna (4.4 kb) + 29.7 μl POP(1X) = 30μl dna. 4.4kb dna (1:100).

Procedure

As of attempt 1.

Results

So-far very exciting, tethers every where, and good tethers. I checked both the samples; 1.1kb and 4.4kb and surprisingly i found plenty of tethers every where. So tethering is successful. I also got successful over stretching with 1.1 and 4.4kb. This is the second time i got overstretching successful. I tried different loading rates and bead heights. The overstretching proves that we have enough stiffness in our trap and our tweezers is ready to unzip.(sb stands for 1.1kb and bb stands for 4.4kb dna) [7]

Attempt .14(feb/08/2011)

I used Ant's old dna (07/15/09) of 1.1 and 4.4 kb. Sane construct as of last attempt.

Components

1μl .5 μm bead + 45μl BGB =50μl (1:100).
.3 μl dna (1.1kb) + 29.7 μl POP(1X) = 30μl dna. 1.1kb dna (1:100).
.3 μl dna (4.4 kb) + 29.7 μl POP(1X) = 30μl dna. 4.4kb dna (1:100).

Procedure

As of attempt 1.

Results

Very successful tethering and overstretching. This time's overstretching proves that it was not one time thing. There are few things i notices; If i keep the laser spot for over some time (30 sec and over)over the the tethered bead, than it looks like it weakens the tether. So we need a very good on/off shutter, which will help us controlling the exposure. And also as you will see some of the overstretching profiles are not at horizontal line, so there might be a power drift again due to the AOM, i will look more into it next time. (sdna stands for 1.1kb and Ldna stands for 4.4kb dna.)[8]

Attempt .15(feb/16/2011)

successful attempt tethering and overstretching wise. I used Ant's old dna (07/15/09)4.4 kb and newly made PCR-dna(2/11/2011) of 55nM concentration, both are stretching ds-dna.

Components

1μl .5 μm bead + 45μl BGB =50μl (1:100).
.1 μl dna (4.4kb; old) + 9.9 μl POP(1X) = 10μl dna of (1:100).
1 μl dna (4.4 kb; new) + 9 μl POP(1X) = 10μl dna of (1:10).

Procedure

As of attempt 1.

Results

Very successful tethering and overstretching. Tethers are every where: {{#widget:YouTube|id=XDQkC9saRjw|600px}}

Tethers are easily found in both the samples. Tethers and overstretching in the new dna-sample proves that the PCR Ant ran, worked. And we are set for unzipping. In some of the overstretching profiles we see the profile on a slope due to the sum signal change at QPD. This problem was due to the ND filter being very close to QPD, as ND filter was moved out, the problem is solved. Another problem was the AOM oscillations, which is due to the crystal, as the laser power modulated by some external source in-front of the AOM-crystal. Due to that I moved the newly constructed shutter after the AOM. So at this point we have solved all the major problems of signal drift at QPD. There is a little momentarily drift because of eclipsing ND filters in-front of the steering assembly first lens, but this is not a problem since we take the data without having that ND filter in way. [9]

Attempt .16(feb/17/2011)

successful attempt of tethering in stretching-dna but not in unzipping-dna. I used Ant's newly-made PCR-stretching dna (TPAL) (02/11/2011)4.4 kb of 55nM concentration. And newly-made PCR-unzipping dna (TPBR)(2/13/2011).

Components

5μl .5 μm bead + 45μl BGB =50μl (1:10).
.1 μl dna (4.4 kb; stretching) + 9.9 μl POP(1X) = 10μl dna of (1:100).
.1 μl dna (4.4 kb; unzipping) + 9.9 μl POP(1X) = 10μl dna of (1:100).

Procedure

As of attempt 1.

Results

Tethering and overstretching was successful in the stretching dna. But there were no tethers in the unzipping dna. [10] [11]

Attempt .17(feb/18/2011)

I tried unzipping dna and stretching dna with T4 dna-ligase protein. I flow protein in the end, in the stretching chamber just to see if protein makes any difference in stretching profiles. Unzipping 4.4kb dna (TPBR; 3.9ng/ml) with .5 μm and stretching 4.4kb dna (55nml) with T4 dna-ligase and with 1μm beads.

Components

.1μl .5 μm bead + 9.9μl BGB =10μl (1:100).
.1μl 1 μm bead + 9.9μl BGB =10μl (1:100).
(.1 μl dna (4.4 kb; stretching) + 9.9 μl POP(1X) = 10μl dna of (1:100)) + (1 μl of T4 dna-ligase + 9 μl POP(1X) = 10 μl protein (1:10)
1 μl dna (4.4 kb; unzipping) + 9 μl POP(1X) = 10μl dna of (1:10).

Procedure

Flow anti-dig 10μl each in both chambers, wait for 6 min.
Flow BGB 50μl each in both chambers, wait for 2 min.
Flow unzipping-dna 10μl in one chamber and stretching-dna 10μl in other chamber wait for 14 min.
Flow BGB 50μl each in both chambers, wait none.
Flow micro-spheres .5μm in unzipping and 1μm in stretching-dna, chamber 10μl each, wait for 14 min.
Flow BGB 50μl each in both chambers, wait none.
Flow protein T4 dna-ligase 10μl only in stretching dna chamber, wait for 10 min.
Seal it.

Results

The things i noticed:

no tethers in unzipping sample.
A new kind of overstretching profile is seen: I called it stair-overstretching. Because you can see stairs; these stairs can be due to multiple tethers or may be due to protein i am not sure yet.
In simple overstretching profiles i noticed a dip in the top flat region of overstretching. This dip is usually seen between 1200 to 1300nms in other words at 1/3 of the length from the right. (see results for the detail and read headings). But the position of the dip does not remain same; in some profiles it is at 1/2 OR 2/3 of the length.
In stretching-dna sample it was not suppose to unzip, but i got one possible unzipping.

[12]

Attempt .18(feb/21/2011)

I tried unzipping 4.4kb dna (UPALS; 5nml) and stretching dna (TPAL;55nml) with .5μm beads. Tethering was successful in both the samples.

Components

.2μl .5 μm bead + 19.8μl BGB =50μl (1:100).
.1 μl dna (4.4 kb; stretching) + 9.9 μl POP(1X) = 10μl dna of (1:100).
1 μl dna (4.4 kb; unzipping) + 9μl POP(1X) = 10μl dna of (1:10).

Procedure

As of attempt 1.

Results

[13]

Tethering was successful in both the samples. There were more tethers in stretching sample.
No unzipping was seen in the unzipping sample. Unzipping sample gave some stretching which looked just like stretching sample.
After couple of hours good tethers are hard to find.
Dip and stair-overstretching was seen again in some profiles.
FTC works but gives a huge offset due to the beam deflection caused by heating of ND-filters.

Attempt .19(feb/23/2011)

I tried stretching 4.4kb dna (UPALS; 5nml) and Unzipping 1.1kb dna (UPBR;55nml) with .5μm beads. Tethering was successful in both the samples but there was more stuck beads than usual. To make these samples I used10μl αPEM.

Components

.5μl .5 μm bead + 19.5μl (9.5μl BGB + 10μl RPEM)=20μl (1:40).
.1 μl dna (4.4 kb; stretching) + 9.9 μl POP(1X) = 10μl dna of (1:100).
1.1 kb; unzipping dna (3.9 ng/mol)

Sample1

This sample had dual chamber with 4.4kb stretching and 4.4kb UpAls unzipping dna. Stretching dna had 1:100 and unzipping had 1:20 concentrations.

Components

10μl .5(1:20)μm bead + 10μl BGB= 20μl (1:40).

1 μl dna (4.4 kb; stretching;TpAls) + 9 μl POP(1X) = 10μl dna of (1:10)==>.

==>1 μl dna (4.4 kb; stretching;TpAls; with 1:10) + 9 μl POP(1X) = 10 μl dna of (1:100).

.5 μl dna (4.4 kb; unzipping; UpAls)+ 9.5 μl POP(1X) = 10μl dna of (1:20).

Procedure

As of attempt 1.

Results

There were no tethers in any of the samples. I think the tape had a leakage because there were big air bubbles in the cambers.

Sample2

This sample also had dual chamber with 4.4kb stretching and 4.4kb UpAls unzipping dna. Stretching dna had 1:10 and unzipping had 1:10 concentrations.

Components

10μl .5(1:20)μm bead + 10μl BGB= 20μl (1:40).

1 μl dna (4.4 kb; stretching;TpAls) + 9 μl POP(1X) = 10μl dna of (1:10).

1 μl dna (4.4 kb; unzipping; UpAls)+ 9 μl POP(1X) = 10μl dna of (1:10).

Procedure

As of attempt 1.

Results

I used higher concentrations this time, to check if the low concentration of dna was the cause of having no tethers in the previous sample. And it looks like that it was. Because there were tethers in this sample. Overstretching sample had plenty of tethers and i got a good set of overstretching data with force 40 ~ 50 pN. I was not lucky with unzipping sample, it was very hard to find tethers; i found 2 to 3 good tethers. But they did not unzip, besides they overstretched in a weird manner: The overstretching force was little low and the profile was not exact.

Attempt .24 (apr/19/2011)

I tried stretching 4.4kb dna (new; 02/11/11) and Unzipping 4.4kb dna (new; 1nmol; 4/15/11) with .5μm beads. Tethering was successful in the stretching sample only.

Components

10μl .5(1:20)μm bead + 10μl BGB= 20μl (1:40).
1 μl dna (4.4 kb; stretching;TpAls) + 9 μl POP(1X) = 10μl dna of (1:10).
1 μl dna (4.4 kb; unzipping;1nmol; UpAls)+ 9 μl POP(1X)=10μl dna of (1:10).

Procedure

As of attempt 1.

Results

There was no unzipping but stretching was successful again. I think used too-much dna in stretching sample. But over stretching was successful. The filters (noise reduction and intensity modulation) are performing excellently.