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

Introduction

This page contains all the experiments reagarding DNA-tethering, overstretching and unzipping.

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More on Optical Tweezers [1]

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 [2]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[3]

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

Results

1μM BEAD.[4]

{{#widget:YouTube|id=OvuG6Ju77h0}} {{#widget:YouTube|id=A_CUeRuwxco}} {{#widget:YouTube|id=ehFeJ165Tis}}

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[5]

1. Flow anti-dig 10μl wait for 5 min.
2. Flow BGB 50μl wait for 2 min.
3. Flow 1.1kb (new)10μl wait for 14 min.
4. Flow BGB wait none.
5. Flow micro-spheres one in each chamber 10μl wait for 14 min.
6. 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. [6]

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: [7].

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:

1. 2X ===> 296.5 μl pop + 3.5 μl NaCl (4molar stock) = 300 ul POP buffer.
2. 4X ===> 288.8 μl pop + 11.25 μl NaCl (4molar stock) = 300 ul POP buffer.
3. 6X ===> 281.25 μl pop + 18.75 μl NaCl (4molar stock) = 300 ul POP buffer.
4. 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:

1. 19 μl 2X pop + 1 ul bead (washed stock)= 20μl
2. 19 μl 4X pop + 1 ul bead (washed stock)= 20μl
3. 19 μl 6X pop + 1 ul bead (washed stock)= 20μl
4. 19 μl 10X pop + 1 ul bead (washed stock)= 20μl

• 1.1kb dna with :

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

1. Flow anti-dig 10μl wait for 5 min in both chambers .
2. Flow BGB 50μl wait for 2 min in both chambers .
3. 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.
4. Flow POP 50 μl( 2X or same X concentration POP)one in one and (4X OR other concentration) in second chamber; wait none.
5. 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.
6. 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

1. 9μl 2X pop + 1μl dna = 10 μl (1:10)
2. 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) [8]

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.)[9]

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. [10]

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. [11] [12]

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

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

Results

The things i noticed:

1. no tethers in unzipping sample.
2. 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.
3. 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.
4. In stretching-dna sample it was not suppose to unzip, but i got one possible unzipping.

[13]

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

[14]

1. Tethering was successful in both the samples. There were more tethers in stretching sample.
2. No unzipping was seen in the unzipping sample. Unzipping sample gave some stretching which looked just like stretching sample.
3. After couple of hours good tethers are hard to find.
4. Dip and stair-overstretching was seen again in some profiles.
5. 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)

Procedure

As of attempt 1.

Results

There was no unzipping but stretching was successful again. The only difference i notices between the αPEM and regular buffer use; there were lot more stuck beads in this sample. So i think αPEM might not be suitable for the small beads but just might work right for the big beads.

Attempt .20 (mar/08/2011)

I tried stretching 4.4kb dna (new) and Unzipping 4.4kb dna (new; 1nmol) with .5μm beads. Tethering was successful in both the samples but there was more stuck beads than usual. Tethers were 1 or 2 out of 10 stuck beads.

Components

• 10μl .5(1:20)μm bead + 10μl BGB= 20μl (1:40).
• .1 μl dna (4.4 kb; stretching) + 9.9 μl POP(1X) = 10μl dna of (1:100).
• .1 μl dna (4.4 kb; unzipping;1nmol)+ 9.9 μl POP(1X)=10μl dna of (1:100).

Procedure

As of attempt 1.

Results

There was no unzipping but stretching was successful again. There were more stuck beads than tethers in both the samples, i do not know why, but i will try again. Overstretching was fine; i got some. But i did not get any unzipping; i got some weird profiles though.

Attempt .21 (mar/09/2011)

I tried stretching 4.4kb dna (new) and Unzipping 4.4kb dna (new; 1nmol) with .5μm beads. Tethering was successful in both the samples and bead stucking problem was solved by making new BGB buffer.

Components

• 10μl .5(1:20)μm bead + 10μl BGB= 20μl (1:40).
• .1 μl dna (4.4 kb; stretching) + 9.9 μl POP(1X) = 10μl dna of (1:100).
• .1 μl dna (4.4 kb; unzipping;1nmol)+ 9.9 μl POP(1X)=10μl dna of (1:100).

Procedure

As of attempt 1.

Results

There was no unzipping but stretching was successful again.

Attempt .22 (mar/10/2011)

I tried stretching 4.4kb dna (new) and Unzipping 4.4kb dna (new; 1nmol) with .5μm beads. Tethering was successful in both the samples.

Components

• 10μl .5(1:20)μm bead + 10μl BGB= 20μl (1:40).
• .1 μl dna (4.4 kb; stretching;TpAls) + 9.9 μl POP(1X) = 10μl dna of (1:100).
• 1 μl dna (4.4 kb; unzipping;1nmol; UpAls)+ 9.9 μl POP(1X)=10μl dna of (1:10).

Procedure

As of attempt 1.

Results

There was no unzipping but stretching was successful again. I used too-much dna in unzipping sample so most of the beads were either stuck or multiple tethers, but no unzipping.

Attempt .23 (mar/28/2011)

I made two samples today both the samples used same dna construct:

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. [15]

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. [16]

Attempt .25 (apr/20/2011)

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 Both the samples. I used (1:1) concentration in unzipping sample and it was successful.

Components

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

I did step dilution for stretching dna.

• (1 μl dna (4.4 kb; stretching;TpAls; 110 nmol) + 9 μl POP(1X) = 10μl dna of (1:10))X 10 ==100 μl (1:10)==>.
• 1 μl dna of the above diluted dna (4.4 kb; stretching;TpAls) + 9 μl POP(1X) = 10μl dna of (1:100).
• 5 μl dna (4.4 kb; unzipping;1nmol; UpAls)+ 5 μl POP(1X)=10μl dna of (1:1).

Procedure

As of attempt 1.

Results

Tethering was successful in both the samples. Overstretching sample performed as usual. I did not try much here. In unzipping sample tethering was successful, but tethers were strangely short (not as floppy as stretching. Almost all the successful tethers overstretched, only one possibly unzipped. I do not know how is it possible? I used .008 loading rate. In this sample the stretched tethers did not stretch upto the usual length, there were kinda short. [17]

Steve Koch 08:41, 21 April 2011 (EDT): Can you compare segments 49 and 1 vertically? If unzipping, pattern should be very similar. (Also, very soon I'll show you how to simulate the unzipping. We'll adapt Larry's software, or just rewrite it with you.) I too, still don't understand the overstretching. I also don't understand why tethers would be shorter.

Attempt .26 (apr/21/2011)

I tried stretching 4.4kb dna (new; 02/11/11) and Unzipping 4.4kb dna (old; 1nmol; 02/11/11) with .5μm beads. I did not dilute the unzipping dna i used it raw (1nMol) of 10μl. The stretching was as usual (diluted from stock of 1:10).

Components

• 10μl .5(1:20)μm bead + 10μl BGB= 20μl (1:40).
• 1 μl dna of the above diluted dna (1:10; 4.4 kb; stretching;TpAls) + 9 μl POP(1X) = 10μl dna of (1:100).
• Raw concentrated dna of 1nMol= 10μl.

Procedure

As of attempt 1.

Results

Unzipping sample: i used .0008 loading rate, There were very few tethers. Some of them broke right away, so no unzipping or stretching. Some were too strong, so keeping coming out of the trap even at full power. Only one tether was successful but it over-stretched. A higher number of beads were stuck. Over-stretching sample: There are few tethers (lower in number than usual), more stuck beads (higher than usual). Some tethers were successfully over-stretched.[18]

Attempt .27 (apr/30/2011)

I tried stretching 4.4kb dna (new; 02/11/11) and Unzipping 4.4kb dna (old; 1nmol; 02/15/11; UpALS) with .5μm beads. I did dilute the unzipping dna with (1:1). The stretching was as usual (diluted from stock of 1:10).

Components

• 10μl .5(1:20)μm bead + 10μl BGB= 20μl (1:40).
• 1 μl dna of the above diluted dna (1:10; 4.4 kb; stretching;TpAls) + 9 μl POP(1X) = 10μl dna of (1:100).
• 5μl dna (raw) + 5μl of pop (1x)=10μl (1:1)

Procedure

As of attempt 1.

Results

Overstretching sample was successful; there were easyly find tethers which overstretched.

In unzipping sample there were tethers. Many tethers were overstretched, a few 1 or 2 unzipped. Result[19].

Attempt .28 (May/18/2011)

I tried stretching 4.4kb dna (new; 02/11/11) and Unzipping 1.1kb dna (old; 3.9 ng/mole; 02/15/11; Upbr) with .5μm beads. I did not dilute the unzipping dna. The stretching was as usual (diluted from stock of 1:10). I also made a sample without dna, to check the solute contamination. I used the sample protocol as attempt 1, just spiked the dna part.

Components

• 10μl .5(1:20)μm bead + 10μl BGB= 20μl (1:40).
• 1 μl dna of the above diluted dna (1:10; 4.4 kb; stretching;TpAls) + 9 μl POP(1X) = 10μl dna of (1:100).
• 10μl dna (raw) without any dilution=10μl
• no dna just beads:10μl of .5μm bead (1:20)+ 10 μl BGB = 20 μl of bead (1:40)

Procedure

As of attempt 1.

Results

In no dna sample, there were no tethers at all, so no sign of solute contamination. But most of the ~95% beads were stuck, i do not know why? Result[20]

Attempt .29 (May/28/2011)

I tried stretching 4.4kb dna (new; 02/11/11) and Unzipping 1.1kb dna (new; 1.4 nmole; 05/25/11; Upbr) with .5μm beads. I did dilute the unzipping dna with 1:5 ratio. The stretching was as usual (diluted from stock of 1:10). I also made a sample for just stuck beads (1 and .5 μm).

Components

• 10μl .5(1:20)μm bead + 10μl BGB= 20μl (1:40).
• 1 μl dna of the above diluted dna (1:10; 4.4 kb; stretching;TpAls) + 9 μl POP(1X) = 10μl dna of (1:100).
• 2μl dna Upbr unzipping dna + 8μl pop (1X)= 10μl of dna (1:5)
• Stuck beads: 5μl of .5μm bead (1:20)+ 5μl of 1μm bead (1:100) = 10 μl of bead.

Procedure

As of attempt 1. stuck bead: # Flow 10μl of NaCl and wait for 5 minutes.

1. flow 10μl of bead wait for 5 minutes.
2. flow 50μl BGB and seal the sample.

Results

First the stuck bead sample; surprisingly no stuck bead, which means that the above protocol did not work. In overstretching sample the tethering and overstretching was usual. In unzipping sample there were very very few tethers, no unzipping. Result[21]

Attempt .30 (June/02/2011)

I made overstretching 4.4kb dna (new; 02/11/11, Unzipping 1.1kb dna (new; 2.4 nmole; 06/02/11; Upbr), and Unzipping 1.1kb dna (1.4 nmole; 05/25/11; Upbr) with .5μm beads. I did dilute the new-unzipping dna with 1:5 ratio and other unzipping dna 1:1 ratio. The stretching was as usual (diluted from stock of 1:10). I also made a sample for just stuck beads (1 and .5 μm).

Components

• 15μl .5(1:20)μm bead + 15μl BGB= 30μl (1:40).
• 1 μl dna of the above diluted dna (1:10; 4.4 kb; stretching;TpAls) + 9 μl POP(1X) = 10μl dna of (1:100).
• 2μl of upbr new-unzipping dna (2.4 nmole; 1.1kb) + 8μl of pop (1X)= 10 μl of dna (1:5)
• 5μl of upbr unzipping dna (1.4 nmole; 1.1kb) + 5μl of pop (1X)= 10 μl of dna (1:1)
• Stuck beads: 2.5μl of .5μm bead (1:20)+ 2.5μl of 1μm bead (1:100)+ 15μl of NaCl (4 moler) = 20 μl of bead.

Procedure

As of attempt 1.

stuck bead:

1. Flow 20μl of stuck bead and seal.

Results

The stuck bead sample was successful, i used it to level the z-piezo and align the camera and trap center. I also used it for DOG and stiffness calibration.

The over-stretching was also successful; it was as usual. I got a set of good over-stretching. Now more than 90% of the beam path is enclosed inside the plastic and foam pipes, which definitely makes a difference in noise. I noticed it.

In unzipping sample there were some tethers. Most of the tethers did no break, stretched or unzipped. A few tethers broke. Result[22][23]

Attempt .31 (June/06/2011)

We prepared new BGB, anti-dig and a new stock .5 μm beads. Dr. Koch prepared a dual-sample of overstretching dna and unzipping dna.

• BGB preparation steps:

1. Weight 200 mg BGM.
2. Mix 20 ml of pop (1X)= 220 ml of BGB solution (10mg/ml).
3. Mix the BGB very well.
4. Now take 750 μl of above solution + 750 μl of pop (1X) = 1500 μl of 1X-BGB solution (5mg/ml)
5. Now filter it and store it in the small tube for sample making. The left over solution is stored in the freeze for later use.

• .5μm bead (Bangs;lot#8892, cat#CPO1F) preparation steps:

1. Shake the bottle to mix the beads.
2. Take our 100 μl from the bottle. This is the stock; keep it at 4 degree centigrade.
3. 5 μl of beads + 45 μl of pop (1X) = 50 μl of bead (1:10) for sample

• Anti-dig preparation steps:

1. 20 μl of anti-dif from the freezer + 180 μl of PBS = 200 μl of anti-dig solution (1:10)

Components

• 30 μl of .5 ml bead (1;10.
• 2 μl dna of the above diluted dna (1:10; 4.4 kb; stretching;TpAls) + 18 μl POP(1X) = 20μl dna of (1:100).
• 5μl of upbr new-unzipping dna (2.4 nmole; 1.1kb) + 10μl of pop (1X)= 12 μl of dna (1:2)

Procedure

1. Clean glass:

Cleaned using just water and scrubbing, followed by water rinse about 6 times. My intuition is that this did not clean the glass well at all. The tape would not stick to the glass, way worse than I've ever seen it. Subsequently, both samples leaked. Sample may or may not

1. Flow anti-dig 10~15μl wait for 4 min.
2. Flow BGB 50μl 3 times into each chamber and wait for 2 min.
3. Flow unzipping and overstretching dna 10~15μl into separate chambers and wait for 5 min.
4. Flow BGB 50μl into each chamber and wait none.
5. Flow micro-spheres 10~15μl one in each chamber wait for 10 min.
6. Flow POP (1X) 50μl into each chamber + seal it with nail polish

Results

In overstretching sample there were plenty of tethers, it was due to the effect of higher bead concentration (1:10 in comparison to 1:40. I did overstretching with .00001 and .0001 loading rates.

In unzipping sample we found many tethers, so tethering was successful in both the samples. Most of the tethers either broke or behaved like a wandering stuck bead. Few tethers stretched also but broke at the point of overstretching. The points to be noted for this experiment are;

• Clean the slides and cover slips with distil-water/ air dry OR with Windex few times before use.
• Flow pop (1X) in the end at the place of BGB. Result[24][25]

Attempt .32 (June/07/2011)

I prepared a dual-chamber sample with overstretching and unzipping dna. overstretching dna is the same as usual with 1:100 concentration. For unzipping i used old 4.7kb UpALS dna with 1:3 concentration. I am using distil water/air cleaned slides.

Sample1.

This sample is dual; created with 1:3 concentration of unzipping dna and 1:100 of overstretching dna.

Components

• 24 μl of .5 ml bead (1:10) from the stock. Stock: 5μl of .5μm bead (out of the bottle) + 45 μl of BGB (1X) = 50 μl of bead (1:10).
• 1 μl dna of the above diluted dna (1:10; 4.4 kb; stretching;TpAls) + 9 μl POP(1X) = 10μl of overstretching dna of (1:100.
• 4μl of UpALS unzipping dna (~1 nmole; 4.4kb; 03/10/11) + 8μl of pop (1X)= 12 μl of unzipping dna (1:3).

Procedure

First cleaning the slides.

Cleaning the slides

1. Scrub the slide with Windex[26].
2. Air dry.
3. Scrub the slide with distil water.
4. Air dry.
5. Repeat the above 4-steps 2 more times.

Sample making

This procedure is slightly changed from attempt 1.

1. Flow anti-dig 10~12μl into each chamber, wait for 6 min.
2. Flow BGB twice 50μl into each chamber, wait for 3 min.
3. Flow DNA 10~12μl unzipping and overstretching into each separate chamber, wait for 7 min.
4. Flow BGB 50μl into each camber, wait none.
5. Flow micro-spheres 10~12μl into each chamber, wait 12 min.
6. Flow pop (1X) into each chamber and seal. (I did not use BGB in this step)

Results

Great result tethering wise; both samples are successful. I did not look much into the overstretching sample. In unzipping sample tethering was good. As an example this video shows a good tether in unzipping sample:{{#widget:YouTube|id=9O4b-2yQzo4}} Look in the blue circle, you will see a tethered bead. Different tethers behaved differently;some overstretched, some broke and a very of them unzipped. I distributed the data in the following categories and the required conditions are given in the bracket:

Data Guidelines

• Total attempts: shows the total number of times i try the same or a different tether.
• Tether-broke: Number of times the tried-tether breaks(when the breaking-force is less than 20pN and tether breaks abruptly).
• Tether-overstretch: Number of times the tried-tether overstretches (when force went above 30pN and tether overstretches or breaks abruptly).
• Tether-unzip: Number of times the tried-tether unzips (when tether gives unzipping profile around 12 to 20pN, with no precalibration).
• Tether-stuck: Number of times bead behaves as a stuck bead (When the bead gives full or half DOG profile; here bead does not have to be visually stuck, it might look like that it has a tether, but does not overstretch/unzip/break just keep falling-out of the trap).
• No-Result: Number of times neither above given categories is true.

Data-statistics is presented below: {{#widget:Google Spreadsheet |key=0ApjWjFYiQdkfdEU2dlFyM29CUFVJS19kejJncTVGU3c |width=900 |height=300 }} Out of 108 tries only 3 tethers unzipped. results are as follows:[27]. Unzipping segments are 15 of 110607\0418; 47, 54 of 110607\0419.

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Sample2.

This sample is a single chamber, with 1:6 concentration of unzipping dna.

Components

• 15 μl of .5μm bead from the above prepared stock.
• 2μl of UpALS unzipping dna (~1 nmole; 4.4kb; 03/10/11) + 12μl of pop (1X)= 14 μl of unzipping dna (1:6).

Procedure

I did not clean the slides. The sample preparation procedure is similar as above.

Results

Read the data guidelines above for data clarification. The Data-statistics is as follows: {{#widget:Google Spreadsheet |key=0ApjWjFYiQdkfdGVuOUppQ3VqNUdrd05zc192Ymp3S2c |width=900 |height=300 }} Out of 120 tries there are 3 unzipping. All the results are presented in this link:[28] Unzipping segments are 51,57 and 60 of 110607.

Attempt .33 (June/14/2011)

In this experiment i tried to tether the anchor-dna piece, to troubleshoot if it is in correct form (ligation wise).

dna-anchor piece

The biotin molecule is 5 base-pairs away from the nick. I made a dual chamber of overstretching dna and 1.1kb anchor piece.

Components

• 3μl of .5μm bead + 27μl of BGB= 30μl of bead.
• 1μl dna of the above diluted dna (1:10; 4.4 kb; overstretching;TpAls) + 9 μl POP(1X) = 10μl of overstretching dna of (1:100).
• 2μl of anchor(5'-bio; 1.1kb; 06/14/11) + 18μl of pop (1X)= 20μl of anchor (1:10).

take above 2μl of anchor(just made, 1:10)+ 18μl of pop (1X)=20μl of anchor (1:100).(use this)

Procedure

Procedure is same as attempt 32.

Result

Results are not very exciting and far from expected. Under expectation the tethers should break as they are pulled under 20pN force, if careful enough should see a small tale of 25 base-pairs. But tethers behave differently; out of 97 11 tethers overstretched, 39 stuck, 46 no-result and 1 looked like broke in the right range. So only ~1% tethers might have the expected form. Data-statistics: {{#widget:Google Spreadsheet |key=0ApjWjFYiQdkfdGRKMmZ1WmwyM1pxX0tIUXNWQkFBZGc |width=900 |height=300 }}

Results:[29].Segment 20 of 110614\0425 is in the 1%, i am not sure about the force though, its little high and the tale length is about 20nm. In data-file 110614\0424 segment 69 onwards is the overstretching sample.

Attempt .34 (June/15/2011)

In this experiment i made 3 different samples; sample 1 is dual chamber with 5'-bio anchor-dna(1.1kb)and int-bio anchor-dna (1,1kb internal biotin BPRL; biotin is located far away from the nick), sample 2 is mono chamber just the anchor piece of 1.1kb (BPRL) without biotin adapter and sample 3 is dual chamber with 5'-bio anchor-dna (4.4kb length in which biotin molecule is located 5 base-pairs away from the nick) and overstretching 4.4kb dna for tethering, to troubleshoot if these are in correct form (ligation wise).

dna-anchor piece

.

Sample 1

This is dual chamber with 5'-bio anchor-dna(1.1kb)and int-bio anchor-dna (1,1kb internal biotin; biotin is located far away from the nick)

Components

• 2μl of .5μm bead + 18μl of BGB= 20μl of bead (1:10).
• 2μl of 5'-bio anchor(1:10; 1.1 kb; 06/14/11) + 18μl POP(1X) = 20μl of 5'-bio anchor dna of (1:100).
• 2μl of int-bio anchor(1:10; 1.1kb; 06/14/11) + 18μl of pop (1X)= 20μl of int-bio anchor (1:100).

Procedure

Similar as attempt 32.

Result

According to my expectations in a right profile, tether should break abruptly at the force less than 20pN. If careful enough i should see a small unzipped tail in 5'-bio and int-bio anchor. In int-bio anchor (1.1kb) sample out of 83 trials 6 are overstretching, no abrupt breaking. Most of the tethers behaved like a tethered-stuck bead. In 5'-bio anchor 1.1kb sample has the same story (for clearance see section 32.1.3 for data-guidance).Result [30]

Sample 2

This is mono chamber just the anchor piece of 1.1kb (BPRL) construct, without biotin adapter.

Components

• 2μl of .5μm bead + 18μl of BGB= 20μl of bead (1:10).

To dilute the anchor-dna, i used step dilution method.

• 1μl of anchor(BPRL; 1.1 kb; 06/14/11) + 9μl POP(1X) = 10μl of anchor dna of (1:10)

1 μl of previous step anchor(1:10; 1.1kb; 06/14/11) + 9μl of pop (1X)= 10μl of anchor (1:100)

1 μl of previous step anchor(1:100; 1.1kb; 06/14/11) + 9μl of pop (1X)= 10μl of anchor (1:1000).

Procedure

Procedure is same as attempt 32.

Result

The purpose behind this experiment is to check if it tethers (means if the biotin is at the wrong place). Good news there were no tethers in the sample.

Sample 3

This is a dual chamber with 5'-bio anchor-dna (4.4kb length in which biotin molecule is located 5 base-pairs away from the nick) and overstretching 4.4kb dna (to check if the tethering works in both the chambers).

Components

• 2μl of .5μm bead + 18μl of BGB= 20μl of bead (1:10).
• 1μl of overstretching dna(1:10 from the stock; TpAls; 110ng/ml; 4.4 kb; 02/11/11) + 9μl POP(1X) = 10μl of overstretching dna(1:100).
• 1μl of 5'-bio anchor(4.4kb; 06/15/11) + 9μl of pop (1X)= 10μl of 5'-bio anchor dna (1:10).

Procedure

Procedure is same as attempt 32.

Result

Tethering was successful in both the samples. My expectations are the same with 5'-bio anchor sample; in the right profile the tether should break abruptly with/without a tale (no longer then 20nm) under 20pN force and it should happen over and over for at least above 50% tethers. Less then 50 % tethers might do rest. If the dna construct is the appropriate one than this must be true.

Data-statistics shows that out of 145 trials, only 6 broke abruptly under the 20pN force. 72 tethers overstretched (to define overstretching here look section 32.1.3) and 6 unzipped. I am not sure what is going on, because ideally the anchor dna doesn't suppose to overstretch or unzip.

Data-Statistics: {{#widget:Google Spreadsheet |key=0ApjWjFYiQdkfdHJkRUJCTEVFYVExX0UyeVRlSWphWmc |width=900 |height=200 }} Result[31] the relevant data is in section 110615\0429 and 0430.

Attempt .35(June/16/2011)

In the experiment i made a dual-chamber sample with int-bio anchor 4.4kb and BMAPS-anchor 4.4kb for the same reasons; to check the construct is right(ligation wise). If the construct behave as expected, it is all good (means if a tether breaks abruptly below 20pN force with a tail(probably not more than 20nm).

Components

• 2μl of .5μm bead + 18μl of BGB= 20μl of bead (1:10).
• .1μl of int-bio anchor(4.4kb; 06/15/11) + 9.9μl of pop (1X)= 10μl of int-bio anchor dna (1:100).
• .1μl of anchor dna (BPALS; 4.4kb; 06/15/11) + 9.9μl of pop (1X)= 10μl of anchor dna (1:100).

Procedure

Similar procedure to attempt 32.

Result

Tethering was successful in the int-bio anchor sample, but there were no tethers in the anchor (no biotin adapter) sample as expected. Out of all the data only segment 27 of 110616\0432 looks like have the right profile. Result [32].

Attempt .36 (June/17/2011)

I made this stuck-bead sample to study the noise. There are some weird oscillations in the data plane. I want to study those. The proceeding are given in a separate page.

Components

1μl of .5μm (1:10) bead + 1μl of 1μm bead (1:10) + 12 ul of NaCl = 14 ul of stuck-bead solution (1:140).

Procedure

Just flow the bead solution 10~14μl into the chamber and seal.

Result

The sample is successful with normally distributed beads.

Attempt .37 (July/11/2011)

I tried new 1.1kb unzipping DNA pBR made on today with 4.4kb overstretching DNA in a dual chamber sample. Unfortunately there were no tethers which can unzip in unzipping sample. Since the DNA concentration was to low according to Ant, may be there is no DNA, even though i used it raw without any dilution. Overstretching sample was as usual full of good tether. Surprisingly i got 5 descent unzipping out of few from this sample.

Components

• 3μl of .5μm bead (stock bottle) + 27μl of BGB= 30μl of bead (1:10).
• 1μl of overstretching DNA(110ng/ml; 4.4kb; 02/11/11)from stock (1:10) + 9μl of pop (1X)= 10μl of overstretching DNA (1:100).
• .1.1kb unzipping DNA-pBR (07/11/11) 10μl raw.

Procedure

As attempt 1.

Result

There were few tethers in the unzipping sample, but none of them unzipped, they behaved like a stuck tethered bead, even at the full power. Overstretching sample was as usual, full of good tethers. I overstretched some tethers and some even unzipped. And this time the unzipping profiles were rather long. Data-statistics shows that out of 85 trials, only 1 broke abruptly under the 20pN force. 59 tethers overstretched (to define overstretching here look section 32.1.3) and 7 unzipped out of which 5 are long-ones.

Data-Statistics: {{#widget:Google Spreadsheet |key=0ApjWjFYiQdkfdGdhb3dTTS1NaTd0X1JXYUxDSEw2R1E |width=850 |height=250 }}

Result:[33] Segment:110711\0475\007,022, 110711\0476\018,025 & 026 are good unzipping.

Attempt .38 (July/14/2011)

In this experiment i mada a dual chamber sample with overstretching DAN in .5x pop + H_2O and .5x pop + D_2O buffer. To check if overstretching force is highe in D_2O bufffer.

Components

• 500μl pop (1x) + 500μl H_2O = 1000μl of pop (.5x)H_2O.
• 500μl pop (1x) + 500μl D_2O = 1000μl of pop (.5x)D_2O.
• 3μl of .5micron bead + 27μl of BGB = 30μl of bead.
• 2μl of overstretching DNA(110ng/ml; 4.4kb; 02/11/11)from stock (1:10) + 18μl of pop (1X)= 10μl of overstretching DNA (1:100).

Procedure

1. Flow anti-dig 12μl, wait for 6 min.
2. Flow BGB 50μl-twice, wait for 2 min.
3. Flow dna 10μl, wait for 14 min.
4. Flow BGB 50μl-twice, wait none.
5. Flow micro-spheres .5μm, wait for 14 min.
6. Flow pop (.5x H_2O/D_2O) 50μl-Twice, wait none.
7. Seal it.

Result

Tethering is very successful in both the samples and looks the same. I have some good overstretching profiles, but the oscillations are really annoying me. Once I am back I am going to deal with it BIG-TIME. On average it looks like that the D_2O overstretching force is little higher; I have not compared both the data sets side by side, and also the data is not calibrated. If the experiment is performed after calibration with FTC than I am pretty sure we will get some thing more accurate.

I also get few POPPED-unzipping; popped unzipping means that it happens after a strong dna stretching/overstretching and looks like something is popped off to make it happen.

Result and data: http://kochlab.org/files/data/Kochlab-daq2/rathi.pranav/Shotgun%20DNA%20Mapping/110714/,All%20segments%20report.html

• C:\,Local Data\rathi.pranav\Shotgun DNA Mapping\110714