SBB09Ntbk-DougDensmore

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Doug Densmore

I started my first PCR today. Plus I started my notebook. Here are the protocols I need to follow for PCR.

1.Make an oligo dilution of (after reconstituting the oligo):

 9uL Water
 1uL 100uM oligo

Now I have a 10uM oligo.

2. Set up the following reaction in a PCR tube:

24uL ddH2O
3.3uL 10x Expand Buffer "2"
3.3uL dNTPs (2mM in each)
1uL Oligo 1, 10uM
1uL Oligo 2, 10uM
0.5uL Expand polymerase "1"
0.5uL Template DNA

I did this for both my ChaI and Inv parts.

3. Next I ran my analytic gel with:

3uL of PCR product
1uL of loading buffer

I used 5uL of the ladder.

After examining the gels, I found that the Inv did not work. Therefore, I re-did the PCR for Inv. However I was able to continue with ChaI.

4. Regular Zymo Cleanup

  1. Add 180 uL of Zymo ADB buffer (brown bottle) to a 33uL or 50uL reaction.
  2. Transfer into the Zymo column (small clear guys)
  3. spin through, discard waste.
  4. Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol)
  5. spin through, discard waste.
  6. Add 200 uL of PE or Zymo Wash buffer
  7. spin through, discard waste.
  8. spin for 90 seconds, full speed to dry.
  9. elute with water into a fresh Eppendorf tube, use the same volume of water as the volume of the original reaction


This process went fine so I then continued with the digest.

5. EcoRI/BamHI Digest

  • Set up the following reaction:
 8uL of eluted PCR product
 1uL of NEB Buffer 2
 0.5uL EcoRI
 0.5uL BamHI
  • Incubate at 37 degrees on the thermocycler for 1hr
  • Run an agarose gel, and melt with 600uL ADB buffer at 55 degrees. ****NOTE: If you are running short of time, this is an acceptable stopping point

Finally I did a gel purify.

6. Zymo Gel Purify

  1. cut out bands minimizing extra gel matter.
  2. put in ependorf tube and add 600uL of Zymo ADB buffer (brown bottle).
  3. heat at 55, shake and/or vortex until the gel has dissolved.
  4. If the DNA is <300bp add 250uL of isopropanol
  5. transfer into the Zymo column inside a collection tube (small clear guys)
  6. spin through, discard waste.
  7. Add 200 uL of PE buffer (which is basically 70% ethanol)
  8. spin through, discard waste.
  9. Add 200 uL of PE buffer
  10. spin through, discard waste.
  11. spin for 90 seconds, full speed to dry.
  12. elute with 8.5 uL of water into a fresh Eppendorf tube

This was all I had time for.

Douglas Densmore 18:52, 2 March 2009 (EST)

Today I came back from my trip to Seattle and picked up where I left off.

Therefore I had to both begin with my two PCR attempts with Inv (W) and (D) as well as start with my ChaI digest.

Starting with the ChaI digest I did a ligation:

7. Ligation of EcoRI/BamHI digests

  • Set up the following reaction:
 6.5uL ddH2O
 1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
 1uL Vector digest
 1uL Insert digest
 0.5uL T4 DNA Ligase
  • Pound upside down on the bench to mix
  • Give it a quick spin to send it back to the bottom of the tube
  • Incubate on the benchtop for 30min
  • Put on ice and proceed to the transformation

After this I transformed this:

8. Transformation by heat shock Competent cells are stored as 200uL aliquots in the -80 freezer as a communal stock.

  1. Thaw a 200 uL aliquot of cells on ice
  2. Add 50 uL of water
  3. Add 30 uL of KCM salts
  4. Put your ligation mixture on ice, let it cool a minute or two
  5. Add 75 uL of the cell cocktail to the ligation, pipette up and down gently to mix
  6. Let sit on ice for 10 min
  7. Heat shock for 2 min at 42
  8. Put back on ice for 1 min
  9. For ampicillin selection, you can plate immediately, otherwise:
 10. Add 100uL of LB, let shake in the 37 degree incubator for 40 min
 11. Plate on selective antibiotics, let incubate overnight

I was able to skip steps 1-4 since the mixture was made. At this point I was able to stop with ChaI for the day.

The second piece was for me to get at least to the Zymo Clean up of the two PCR reactions for Inv. I was able to do this. I will have to go into the lab tomorrow to transform this.

Douglas Densmore 17:32, 3 March 2009 (EST)

Today I came into the lab outside of class. I had two tasks to accomplish.

1. Take the my Inv part (which had only gone through the first zymo cleanup) all the way through to transformation. I had to do this for my (W) and (D) PCR products (just to be safe).

W-water

D-DSMO - makes the PCR more "promiscuous" (helps DNA denature)

The first thing I need to do was an EcoRI/BamHI digest of my PCR product.

The second step was a gel purify.

The next step was to ligate/transform/rescue and plate. This is where I will leave this part until tomorrow.


2. Take my ChaI part which had been transformed and plated and pick my colonies.

For this I followed the following:

  • For each construct you will pick and later miniprep 2 colonies
  • Add 4mL of LB media with the appropriate antibiotics to a clean test tube
  • Pick a well-isolated, round, and "normal" looking colony with a toothpick
  • Drop it in the test tube
  • Incubate at 37 overnight

A couple of things to note. The first is that this protocol does not mention that you should do this by an open flame (Tim told me this). Also secondly in my case the antibiotics that I needed were AK.

Since these have to incubate overnight. This is all I could do.

Random stuff I want to write down so that I can remember it: The steps after you have a cleaned, purified part are:

  • Ligate
  • Transform (heat shock)
  • Rescue - warm and shake
  • Plate
  • Pick a colony
  • Miniprep.

Douglas Densmore 15:36, 4 March 2009 (EST)

Ok yesterday I did not really take the Inv parts all the way to transformation. Today I did this. I had to 1. ligate my gel purified digestion 2. then transform/rescue/plate.

I was able to to all of these steps today. This required a CA antibiotic plate.

For my second part, ChaI I was able to take the colonies I had picked and do a mini prep. The mini prep procedure was:

  1. Pellet around 1.5 mL or 2 mL saturated culture by spinning full speed, 30 seconds.
  2. Dump supernatant, repeat to pellet another 1.5 mL (for a total of 3 mL)
  3. Add 250uL of P1 buffer into each tube. Resuspend the cells using a vortexer.
  4. Add 250uL of P2 buffer (a base that denatures everything and causes cells to lyse). Gently mix up and down. Solution should become clearer.
  5. Add 350uL of N3 buffer (an acid of pH ~5 that causes cell junk - including protein and chromosomal DNA - to precipitate, and leaves plasmids and other small molecules in solution). Slowly invert a few times, then shake.
  6. Spin in centrifuge at top speed for 5 minutes.
  7. Label blue columns with an alcohol-resistant lab pen.
  8. Pour liquid into columns, and place the columns into the centrifuge. Spin at 12000 rpm for 30 seconds.
  9. Dump liquid out of the collectors under the columns (the DNA should be stuck to the white resin)
  10. Wash each column with 500 uL of PB buffer.
  11. Spin in centrifuge at 12000rpm for approximately 15 seconds, then flick out the liquid again.
  12. Wash with 750uL of PE buffer (washes the salts off the resins).
  13. Spin in centrifuge at 12000rpm for approximately 15 seconds and flick out liquid again.
  14. Spin in centrifuge at full speed for 1 minute to dry off all water and ethanol.
  15. Label new tubes and put columns in them.
  16. Elute them by squirting 50uL of water down the middle of the column (don't let it stick to the sides).
  17. Spin in centrifuge at top speed for 30 seconds.
  18. Take out columns and cap the tubes.
  19. Clean up - note the P1 buffer is stored at 4degC and all the rest at room temperature.


I have put the mini prep result in the "green stripe" box with the rest of my intermediate results. I am going to now be gone for a week. When I get back I will be picking a colony for my Inv part and doing mapping for my ChaI part.

I really need to put some some cool pictures of my gels on here. I guess I should also clean up my notebook as well but I guess at least I am putting things here:)

Douglas Densmore 13:40, 16 March 2009 (EDT)

Now that I am back it is time for mapping ChaI and figuring out what is going on with my Inv part. Someone by mistake filled in that I had no colonies for my Inv part. This is not true. There were colonies.

Anyway, the first thing I am going to do is take care of mapping my ChaI part. Here are the steps:

6.5 uL ddH2O
2uL Miniprepped plasmid
1uL 10x NEB Buffer 2
0.5uL EcoRI
0.5uL BamHI (for parts >250bp) or XhoI (for parts <250bp)
  • Incubate at 37 on the thermocycler for 30 minutes
  • Run an analytical gel
  • Take a picture of the gel
  • Calculate the expected fragment sizes
  • Are the calculated sizes consistent with the bands on the gel?


While I was waiting for my ChaI part in the incubator, I picked colonies for Inv(w) and Inv(D) parts that I had. Once they were picked, there was nothing to do but wait to miniprep them tomorrow.

The ChaI part was fine based on the mapping so I gave my miniprepped parts to Chris for sequencing.

Douglas Densmore 15:31, 18 March 2009 (EDT)

Yesterday I miniprepped my Inv parts. Today I mapped them. They were "fine" so I gave this to Chris to enter in the stock spreadsheet. I still have a lot of trouble reading the gel. I have to take Gabe and Tim's word for this 90% of the time. This part is 2593bp long so it requires two oligos (one from each side) and hence two stock numbers.

Hopefully now I am done building my parts:)

Now to look at the mapping results. Today I finally had a chance to look at my mapping results for my ChaI stock which had come back a day earlier. Chris had told me that they were ok but I wanted to look anyway so that I got some practice looking at the calls. This strikes me as something that could be automated in the future (at least to find partial matches).

The first stock part was sb0076. Looking at the results:

Looking at the call for this clone, index 71-468 is a perfect match. This covers the part through the BamHI tail. So the 3' end is fine. The 5' end does not match. There are 46bp in the ChaI part starting from EcoRI which are not found. However based on the fact that this is before position 71 in the call, I do not trust this area anyway based on the trace. I am going to call this a *match*.

My second stock part was sb0076. These results are the same as the other clone. I will call these a match as well. I am going to update the sequencing log to take this into account.

The fact that both clone calls are the same give me more confidence that things are fine.

Douglas Densmore 15:45, 23 March 2009 (EDT)

Here is what I have found looking at the sequencing results for sbb084 and sbb085. Both calls suffer from bases being swapped and in 84 there is an insertion and in 85 there is a dropped base. My initial feeling is that this is not going to be good:( The chances that these mutations are silent is slim and I am sure the reading frame is going to be wrong. I need to investigate this more along with sbb0082 and sbb0083.

sbb0084

Here are the base pair indices in the call:

30-295 - perfect match
c instead of t
297-359 - perfect match
g instead of a
361-369 - perfect match
c instead of t
371-457 - perfect match
c instead of t
459-571 - perfect match
t instead of c
573-723 - perfect match
g inserted in call
725 - 967 - perfect match


sbb0085

(The call is reverse-complemented). Here are the base pair indices in the call:


327-355 - perfect match
dropped a t
356-369 - perfect match
dropped a c
370-402 - perfect match
c instead of tt
404-420 - perfect match
g instead of a
422-555- perfect match

Douglas Densmore 17:59, 24 March 2009 (EDT)

Today I looked at sbb0083. This was a little better than the other two calls examined so far.

sbb0083

Here are the base pair indices in the call:

27-292 - perfect match
c instead of t
294-356 - perfect match
g instead of a
358-366 - perfect match
c instead of t
368-454 - perfect match
c instead of t
456-568 - perfect match
t instead of c
570-720 - perfect match
gg inserted in call
723 - 759 - perfect match

Douglas Densmore 16:27, 25 March 2009 (EDT)

Today I looked at my last clone, sbb0082.

Here are the results of my sequencing analysis:

Reverse complement the call
222-247 - Perfect match
c instead of g
also in this region ac is not in the clone but in Inv
249-410 - Perfect match
c instead of t
412-429 - Perfect match
g instead of a
431-564 - Perfect match
g instead of a
566-793 - Perfect match

I am going to have to talk to Anderson about all these results. Again, I have a feeling that these will not all be silent mutations.

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