McGill/Team 1: Triple Ligation

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May 2007

May 14

1. Made the same as May 15, but did not use Type 1 (milliQ) H2O, so the solutions were discarded.

May 15

1. Made 1M CaCl2, and glycerol/CaCl2 solutions
   1. 1M CaCl2 Solution: 100mL was made
      1. CaCl2 Dihydrate = 146.986 g/mol
      2. 100mL of 1M CaCl2 requires 14.699g CaCl2 Dihydrate
   2. Glycerol/CaCl2 Solution: 30mL was made
      1. 5.00mL of 60% glycerol solution
      2. 3.00mL of 1M CaCl2
      3. 22.00mL of Type 1 (milliQ) H2O

May 16

1. Transformation of Cells:
   1. Transformed 1 vial of cells with Jun vector, and another vial with Fos vector (1uL of vector each); Sterile
   2. Used 400uL of LB (sterile) for each vial
   3. Incubated for 1 hour in shaker at 37C, then incubate overnight at 37C

May 30

1. Test PCR Machine

June 2007

June 4

1. Made Kanamycin plates
   1. 200mL of Agar
   2. 200mL of 2x LB
   3. 2mL of Kanamycin

June 20

Today: extract DNA from plates, transform, plate on amp plates.

I - Extraction of DNA from Plate (5M and 9G)

1. Puncture foil with pippette tip
2. Add 15uL of Type 1 water
3. Remove all liquid (with DNA in solution)

II - Transformation of extracted DNA

1. Chill cells on ice for 10 min
2. Heat shock cells at 42°C for 30 sec
3. Add DNA for cells
4. Add 500uL of 42°C SOC medium to each vial
5. Incubate on ice for 1 min
6. Place in 37°C shaker incubator for 1h 30min
7. Plate onto 4 separate amp plates
   1. 25uL R0062
   2. 25uL C0060
   3. 400uL R0062
   4. 300uL C0060
8. Place into 37°C incubator at 3:55PM

Next: seed, miniprep and screen the DNA.

June 21

Checked colonies in the 37°C incubator. Well formed colonies were on each plate, so plates (all 4) were transferred to the small fridge (middle shelf on the right).

June 22

Today: miniprep DNA from July 20th transformation

I - Miniprep of Colonies from R25, R400, C25, C300 (indication the biobrick and the amount in uL that was plated)

1. Transfer 1.5mL of cells suspended in LB into a microcentrifuge tube.
2. Centrifuge for 2 min at max speed (14000 rpm)
3. Remove supernatant with pipette and discard.
4. Add 250uL of buffer P1 and 250uL of buffer P2
5. Close and invert tubes 6 times
6. Add 350 uL of buffer N3 (within 5 min of adding buffer P2)
7. Close and invert 6 times
8. Centrifuge for 10 min at 13000 rpm
9. Carefully pipette off the supernatant into a Qiaprep spin column. NB: for sample R400 a large amount of supernatant was discarded. No reason, stupid move by Tim.
10. Centrifuge spin column for 60 sec
11. Discard flow-through
12. Add 750uL of buffer PE
13. Centifuge for 60 sec (max speed)
14. Add 50uL of sterile water (of EB buffer could be used)
15. Let stand for 1 min then centrifuge for 1 min
16. Transfer flow-through to a new micro-centrifuge tube (one with a cap) with a pipette.
17. Stored in -20°C freezer (green box labelled "Team 2")
18. Remaining cells in LB stored in small fridge

Next: Screen the DNA.

June 26

Today: Screen of R0062 and C0060 from July 20th transformation

I - For R0062 add:
1.5uL of buffer 2
1.5uL of 10X BSA
3uL mini-prepped DNA
1uL of XmnI
1uL of XbaI
7uL of sterile water
Total volume = 15uL

II - For C0060
Same as above but no XbaI and 8uL water

Let both sit in 37°C water bath for 1h 40 min (From 3:30PM to 5:10PM)

III - Gel electrophoresis

1. Add 12uL of water to each R25, R400, C25, C300
2. Add 3uL of respective digested DNA
3. Add 1.66uL of loading dye
4. Gel contains:
   1. Ladder (5uL)
   2. R25 uncut
   3. R25 cut
   4. R400 uncut
   5. R400 cut
   6. C25 uncut
   7. C25 cut
   8. C300 uncut
   9. C300 cut
5. The remaining uncut mini-prepped DNA was stored in the pink "Team 1" box.

Next: Interpret the screen and proceed to midi-prep or reseed.

June 28

Today: Reseeding of R0062

I - Reseeding of R0062

1. Prepare 6 culture tubes with 2.5mL of sterile water and 2.5 mL of LB medium
2. Add 5uL of 1000X Amp to 5 of the tubes (no antibiotic added to the control)
3. 3 seedings from R25, 2 seedings from R400
4. At 4:00PM the seedings were placed in the 37°C shaker incubator (in styrofoam pieces wedged inbetween clamps.
5. Plates returned to fridge

Next: Miniprep and screen the re-seed.

June 29

Today: Digest and screen the re-seed from June 28th

I - Digest using same procedure as June 26th

1. DNA was removed from the water bath at 4:35PM

II - Gel electrophoresis

1. Gel contains
   1. Ladder (5uL)
   2. 400A
   3. 400B
   4. 25A
   5. 25B
   6. 25C
2. Gel turned on at 5:02PM. 100V set for 40 min with variable current.
3. Picture saved as "June 29 R400A R400B R25ABC"

Next: Interpret the gel and plan appropriate course of action.

July 2007

July 5

Today: Digest and screen R and C from July 4th midiprep

I - Digest Volumes
1.5uL 10X buffer
0.5uL suspended DNA (less than usual b/c midiprep is more conc.)
1uL EcoRI
12uL water
Total volume - 15uL

At 11:52AM tubes were placed in a 26.2°C water bath that was on its way up to 37°C.

II - Spectrophotometric DNA quantification

1. Dilute DNA by a factor of 1000 (i.e. 1uL of DNA in 999uL of water). Mix by tapping.
2. Reference (at 260 nm) a quartz cuvette that has been rinsed and filled with water.
3. Rinse the cuvette with DNA solution.
4. fill the cuvette and take an absorbance reading at 260 nm.

Results: A(R@260nm) = 0.022;    a(C@260nm) = 0.75

Calculation: [DNA] = 50 x (dilution factor) x (Absorbance at 260nm)
Therefore [R] = 1100 ng/uL; [C] = 4250 ng/uL

Next: Gel quantification and ligation

July 6

Today: Gel quatification of "I" and "R"

I - Gel quantification
Wells contain:
7uL water
1uL loading dye
2uL DNA (either the vector "R" or the insert "I")
Order of wells:

1. Ladder (5uL)
2. Insert
3. Vector

The gel was run for 45 minutes.

July 11

Today: Transformation of ligation product (R+C)

I - Transformation

1. Same procedure as June 20 except:
   1. All of ligation rpoduct was put into cell capsules
   2. Heat Shock was 2 min b/c they are Top 10 cells
2. Plates were stored overnight in 37°C incubator.

Next: Seed and miniprep

July 12

Checked plates and there was no growth, so we have to set up a the ligation again.

July 17

Today: Plating of ligation and extra plasmid (to check the efficiency of transformation)

I - Plating

1. Went to take cultures out at 2:30PM but the shaker had been turned off. So the shaker was turned back on and left to run for an additional 15 min.
2. Tubes were removed from SI at 2:45PM and cells were plated on Amp plates.
   1. Control (R400) 25uL
   2. Control (R400) 300uL
   3. Ligation 25uL
   4. Ligation 250uL
3. Stored in 37°C incubator over night

Next: Seed and miniprep

July 18

Today: Seeding of ligation plate (R0062 from July 17)

1. Prepare 10 culture tubes
   1. 2.5mL sterile water
   2. 2.5mL LB medium
   3. 5uL Amp
2. Seeds were only taken from the 25uL ligation plate. The 250uL plate was too full to distinguish colonies.
3. Prepared two controls as well
   1. water and LB only
   2. Water + LB + Amp
4. All tubes were placed in 37°C incubator at 4:05PM

Next: Miniprep and screen

July 19

Today: Miniprep of Ligated DNA (R0062 from July 17)

NOTE: After starting, Alex pointed out that due to the lack of ribosome binding site in our construct, what 
 we are miniprepping is useless.  We will keep going though to make sure that our ligations are working.

I - Miniprep

1. Follow usual mini-prep protocol as per June 22.
2. We had to wait before proceeding to the screening digest because there were no small tips autoclaved. Miniprepped DNA was left on benchtop for the 20 min that tips were autoclaving.

II - Screening Digest 5uL of mini-prepped DNA
1.5uL of buffer 2
1.5ul of 10X BSA
1uL of EcoRI
1uL SpeI
5uL sterile water

Gel contents:

1. Ladder
2. Culture A
3. Culture B
4. Culture C
5. Culture D
6. Culture E
7. Culture F
8. Culture G
   

Cultures H through J, we ran out of SpeI and so were not digested or run on the gel.

Results: We know how to ligate. YAY! Now we restart.

Next: Incorporate a RBS

July 20

Today: Extraction of B0034.

I - Extraction of B0034.

1. Pierce the foil top with a pipette.
2. Extract the DNA with 15ul of sterile water.
3. Transfer to a microcentrifuge labeled [B0034; 20/07/07; 15ul] and stored in pink "Team 1" box.

II - Tansformation

1. Allow Top 10 cells to thaw on ice for 10 min. DNA was also placed in the ice bucket.
2. Add 2uL of DNA to Top 10 cells.
3. Incubate on ice for 30 min then heat shock at 42°C for 30 sec.
4. Add 500uL of LB, 250uL SOC and ice for 1 min.
5. Place in 37°C shaker incubator for 1 hour (started at 3:12PM)
6. Plates put into 37°C incubator at 4:50PM
7. Remaining cells placed in Team 1 freezer box; labelled "B0034 cells"

Next: Seeding

July 21

Today: Seeding of B0034. Hanmo and Allan performed the seeding. Standard protocol. Two tubes per concentration plate (i.e. 25 and 250)
Placed into 37°C SI at 1:20PM

Next: Mini-prep

July 22

Today: Dilution of seeding. We took 250uL of cultured cells from the culture tubes, and diluted each into a flask. Then we added 25mL of 1X LB and 25uL of Amp to each flask. The dilutions were then placed into the shaker-incubator room at 37C overnight.

Next: Mini-prep.

July 23

Today: Midiprep of B0034

The midiprep was done according to Annette's protocol

July 24

Today: Quantification of Midi-prepped B0034 NB: On July 5th we diluted by a factor of 1:1000, but this was because we expected the DNA to be very concentrated (and it was). Depending on the volume of DNA solution (and therefore the supposed concentration)the dilution might only have to be 1:100. But it turns out that due to the barely visible pellet we will proceed with a 1:1000 dilution.

Results: A(B1@260nm)= 0.086 A(B2@260nm)=0.087
[DNA]= 50 x (duilution factor) x (absrobance @ 260nm)
= 150 ng/ul for B1
= 200 ng/ul for B2
In the future we might want to use the 1:100 dilution Next: Sceen the midi-prep and ligate

July 25

Today: Sceening digest of Midi-prepped B0034

Only 0.5ul of Ladder was added because we were using syber-green. the load in other wells was: 1ul DNA, 1.67ul loading dye, 14ul water (total: 16.67ul)
The gel was loaded as:

1. Ladder
2. uncut B0034
3. cut with XbaI
4. cut with XmnI + BseRI
5. cut with BseRI
6. cut with XmnI
7. cut with XmnI + XbaI

The gel was started at 3:36PM and run for 50 min. The gel picture was saved as "July 25 B0034 5 enz". Results show appropriate bands, and presence B0034 is confirmed.

Next: Preparation for ligation of C0060 into B0034.

July 27

Today:

1. Overnight large scale digest of B0034: to open up the plasmid to prepare for ligation
   1. 5ul Buffer 2
   2. 5ul 10X BSA
   3. 5ul "B2" B0034 midiprep DNA
   4. 1ul PstI (75% activity)
   5. 1ul SpeI (100% activity)
   6. 33ul dH2O

July 28

Today:

Turn off water incubator (37C) and placed in the freezer to stop the digest at 12:56PM. The tube is in the pink Team 1 freezer box. (It's the only tube with tape on it)

Next: Ligation

July 30

Today:

1. Heat inactivation: 20 minutes at 80C
2. Ran 2ul of B0034 digest for quantification
3. Set up ligation for B0034 + C0060

July 31

Today: Transformation of B0034+C0060 ligation, and plated it onto 2 plates

1. Plate 1: 70ul cells
2. Plate 2: 215ul cells

August 2007

August 1

Today: Seeding of the B0034+C0060 ligation

5 colonies from the 70ul plate and 5 from the 215ul plate (+2 controls)
Each tube contains:

1. 2.5ml of 2X LB
2. 2.5ml of distilled water
3. 5ul Ampicillin

Culture tubes placed in the 37C shaker incubator at 4:58PM. Plates were put back in the fridge (top shelf right hand side). NB: the 215ul had a cracked lid; the crack has been sealed with tape.

Next: Mini-prep and screening digest

August 2

Today: Mini-prep of seedings from Aug 1

1. Screening digest carried out with EcoRI and SpeI in EcoRI buffer. The mini-prepped DNA was diluted by an extra 5ul of water during the digest so more DNA and less water was used when loading the gel (to ensure a decent resolution).
2. According to the results of the gel, we diluted 250ul of cells in suspension from tube 6f (215ul batch, #5) in 25 ml of LB and 25ul Amp, for Midi-prep.
3. The flask went into the SI (37C) at 6:15PM.

Next: Midi-prep.

August 3

Today: Midi-prep and screen

1. Note that the SI was turned off when Steph checked this morning, but there was some growth, so we will proceed nonetheless. The Midi-prepped DNA was digested in two batches to ensure that everything worked properly.
2. Digest 1: BsrGI and SpeI with expected bands at 2455bp and 425bp.
3. Digest 2: BseRI and SpeI with expected bands at 2096bp and 784bp.
4. Digests were carried out in the 37C water bath starting at 12:23PM

1. We obtained unpromising results from the second digest. We suspect that the SpeI may be faulty, so we will try to cut it with BseRI and PstI. In buffer 2 PstI has only 75% efficiency so we will leave it overnight. Digest was started at 4:09PM.

1. Midiprep quantification: O.D. was 0.001, which is too low.

1. After speaking with Jay we are going to try to regrow some cells left over from the original seeding (the 1 out of 10 that worked). There was about 300ul left. We diluted 250ul in 25ml of LB. Cells went into the incubator at 5:26PM.

Next: Check on new digest and on cells in incubator.

August 4

Today: Check on new digest and on cells in incubator Digesting DNA was taken out of the 37C incubator at 11:08AM. BAD NEWS from the gel. Only one band b/w 3kb and 2kb and it wasn't a short band that ran off the gel, the smallest section of ladder was halfway down the gel. Picture is saved as "Aug 4 (BseRI + PstI)"

Also the bleach bottle is cracked and I didn't have time to switch it.

Next: Check on new digest and on cells in incubator.

August 7

1. Set up new ligation (same amounts as last time)
2. Susan and Avi placed it in Piccirillo lab's 16C thermal block for overnight ligation

August 8

Today: Plating of B0034+C0060 (the second ligation attempt)

1. Put Amp plates in the 37C incibator to warm them up at 2:25PM
2. Plates went into the incubator at 3:23PM. Right Hand wall middle shelf

Next: Seed, mini-prep and screen

August 9

Today: Seeding and prepping reagents
I - [Preparation of 500ml of 2X LB]x2
II - Preparation of 200ml of agar

1. 6g in 200 ml of water

III - 3 x 500ml of sterile water (type 2)
All went into the autoclave on setting 1 at 1:15PM

IV - Seeding of ligation: 250ul plate only

1. Ten cultures (2.5ml LB, 2.5ml water, 5ul amp)
2. Put into 37C shaker at 2:30PM

Note: the 25 ul plate had very abnormal growth so it was not seeded.

Next:mini-prep

August 10

Today:

1. Mini-prep of 10 overnight cultures of ligated DNA
2. Screening Digest of the 10 cultures:
   1. EcoRI + SpeI (2098bp + ~800bp)
   2. BsrG1 (~3000bp)
   3. All 20 digests went into the 37C at 2:10PM
3. Gel electrophoresis:
   1. We used 3ul of digested DNA, 12ul water, 1.66ul of loading dye
      1. Gel 1: double digest
      2. Gel 2: single digest
   2. Results:
      1. Culture 6 showed good results in both gels
      2. Culture 3 showed good results for the double digest but not the single cut
      3. Culture 8 showed good results for the single but not the double
4. We prepared midi-prep dilutions of all three cultures (25ml LB, 250ul cells, 25ul amp) and we will screen the dilutions once the midiprep is complete.
   1. The dilutions went into the incubator at 6:40PM

Next:Midi-prep

August 11

Today:

1. Midi-prep of dilutions of cultures 3, 6 and 8.
2. After midiprep the DNA was quantified spectrophotometrically.
   1. 3: 108 ng/ul
   2. 6: 30 ng/ul
   3. 8: 990 ng/ul
3. Due to the small amount of DNA from #6 we prepared another dilution in case it needs to be re-midiprepped

1. Gel electrophoresis
   1. Culture 8: cut with BsrGI one band at 3000bp
   2. Culture 3: cut with BsrGI one band at 1300bp
   3. Culture 8: cut with EcoRI + SpeI faint band at 3kbp others at 2kbp and 700bp
   4. Culture 3: cut with EcoRI + SpeI one band 2kbp

File saved as "August 11 C+B midi screen digest"

Next: Large scale digest for ligation.

August 12

Today:

1. Midiprep of #6 (2nd attempt)
   1. Quantification: O.D. = 0.076
      1. 50x300x0.076 = 1140 ng/ul
2. Large scale digest of #6 and #8
   1. Midi-prepped DNA (1.5ul)
   2. Xba (1ul)
   3. PstI (1ul)
   4. BSA (5ul)
   5. Buffer 3 (5ul)
   6. 37ul dH2O

Place in 37C water bath at 4:32PM. Will be left overnight because of the large amount of DNA to cut, and XbaI has only 75% efficiency in buffer 3. There is no star activity, so it shouldn't be a problem.

Next: Gel extraction of B0034+C0060

August 13

Today: Gel extraction

1. Running the Gel
   1. Result: 8 was bad, 6 was cut properly

1. Gel extraction
   1. Weigh excised gel pieces
   2. Add 3ul of QG buffer for every 1mg of gel
   3. Put in 50C water bath for 10 min (vortex a bit every 2-3 min)
   4. Solution should be yellow
   5. Add 1 ul of isopropanol for every 1 mg gel
   6. Load into a spin column, spin for 1 min, discard flow-through
   7. Add 0.5ml QG wash, spin 1 min, discard flow-through.
   8. Add 0.75ml PE wash, spin 1 min, discard flow-through.
   9. Spin for 1 more min, discard flow-through
   10. Place column into clean micro-centrifuge tube
   11. Add 30ul EB, let stand 1 min (can also use 50ul of EB, but this will give a mor concentrated solution)
   12. Centrifuge 1 min, do not discard flow-through (it has the DNA in it)

1. Quantification:
   1. O.D. = 0.003: 45 ng/ul
   2. Absolute amount: 5.4 ug

1. Re-Quantification of "cut R" (R0062)
   1. 90 ng/ul

1. Ligation of "R cut" and B0034+C0060
   1. Total volume: 20ul
   2. Conditions:
      1. T4 Ligase (1ul)
      2. T4 buffer (10X) (2ul)
      3. Vector 'R cut' (2.83ul)
      4. Insert B+C (14.16ul)

Went into 16C heat block at 6:38PM

Next: Transformation.

August 14

Today: Transformation of (B+C)+R ligation

1. Transformed 10ul of ligated DNA
2. Two plates: 70ul and 200 ul (used the new plates)
3. Plates went into the 37C incubator at 3:30PM

Next: Seeding

August 15

Today: Seeding of colonies

1. The 70ul plate had better colonies (more distict and larger); 200ul plate had too many tiny colonies
2. As usual: 2.5ml of LB, 2.5ml water, 5ul amp
3. Took 10 colonies from 70ul plate
4. Went into SI at 1:00PM

Note one of the bottles of LB was contaminated.

Next: mini-prep

August 16

Today:

1. Mini-prep of 10 cultures
2. Screening digest:
   1. Single cut: BseRI should give ~3kbp
      1. Conditions: 1.5ul Buffer 2, 1ul BseRI, 5ul DNA, 7.5ul water
   2. Double cut: EcoRI + PstI should give 2.1kbp and ~800bp
      1. Conditions: 1.5ul EcoRI buffer, 1.5 ul BSA, 5ul DNA, 1ul EcoRI, 1ul PstI, 5ul water
3. Gel electrophoresis: No results were good for any of them, so we seeded another 18 colonies from the plate

Next: mini-prep

August 17

Today:

1. Mini-prep of 18 cultures
2. After digesting with BsrGI (it cuts in the insert, and we expected a 3kb band) and running the DNA on a gel we concluded that ligation failed and that all we were plating was recirularized plasmid
   1. Cultures 1-9 on gel 1, and cultures 10-18
   2. Results:
      1. All bands on both gels were at ~1.3kb, which indicates that they were not digested because there was no restriction site available for BsrG1, i.e. the insert was not present.

Conclusion: ligation failed

1. In our next attempt we will adhere more closely to the NEB procedures for T4 DNA ligase

Next: Re-attempt the ligation

August 21

1. As per Elvis's (Jay's graduate student) method
   1. After heat inactivation (20min @ 80C), take a few uL of both insert and vector
   2. Then add dH2O, Ligase, Buffer
   3. Ligation at room temperature
      1. One sample for 4 hours
      2. Other sample for overnight ligation
2. Ligation of R0062 (vector) + B0034+C0060 (insert)
   1. 6ul insert
   2. 4ul vector
   3. 2ul buffer
   4. 1ul ligase
   5. 7ul dH2O
3. Overnight ligation at room temperature (began at 3:50PM)

1. Ligation with Roche "Quick Ligation Kit"

[Insert] = 45ng/ul [Vector] = 90ng/ul

1. 1. Materials:
      1. 0.5ul of Vector
      2. 5ul of Insert
      3. 4.5ul of 1x DNA dilution buffer
      4. 10ul T4 DNA ligation buffer
      5. 1ul T4 DNA ligase
   2. Prepare 1x DNA dilution buffer
      1. 5ul: 1ul of 5x DNA buffer + 4ul type II H2O
   3. Procedure
      1. 2.5ul vector + 5ul insert + 4.5ul of 1x DNA dilution buffer
      2. Mix T4 DNA ligation buffer
      3. Add 1ul T4 DNA ligase
      4. Incubate for 5 minutes @ 15-25C
      5. Analyze via gel

1. Screening digest of Quick ligation
   1. 1ul BseRI
   2. 1.5ul Buffer 2
   3. 1.5ul 10X BSA
   4. 5.0ul DNA
   5. 6.0ul dH2O
   6. Expected Bands: 0.7kb (unligated insert), 2kb (unligated vector), 3kb (cut ligated construct)

1. Gel:
   1. No bands from the ligation at all

Will try to screen the overnight ligation tomorrow to see if it will work

August 22

1. Gel extraction of B0034+C0060 insert just in case we need to set up a ligation again
2. Desktop ligation: as per Elvis's method

Next: Transform the ligated DNA

August 23

Transformed used NEB 5alpha cells

1. Wanted to save time because it was 5AM, so we only put the cells in shaker for 10 minutes.

August 24

1. No growth on plates: bad transformation
2. Another gel extraction of the B0034+C0060 insert
   1. Cannot get any DNA at all: Gel extraction kit may be bad

August 30

Today: Digest of R and B+C

1. Digest of R0062
   1. DNA 2ul
   2. SpeI 1ul
   3. PstI 2ul
   4. Buffer 2 2ul
   5. BSA 2ul
   6. Water 11ul

1. Digest of B0034+C0060
   1. DNA 2ul
   2. XbaI 2ul
   3. PstI 1ul
   4. Buffer 3 2ul
   5. BSA 2ul
   6. Water 11ul

Placed into 37C incubator at 1:32PM

Next: Ligation

August 31

Set up another ligation

September 2007

September 4

Today: Transformation of R+(B+C) into Jay's high efficiency cells

1. Use 3ul DNA ("LIG 4ul 31/08")
2. Cells came out of the -80C at 5:01PM
3. At 5:20PM add 950ul of room temperature SOC medium (we waited longer because the SOC was frozen and had to thaw)
4. Iced for 30 min
5. At 6:32 heat shocked (42C) for 30 sec, then iced for 5 min
6. Add 950 ul SOC (room-temp)
7. Place in 37C SI for 1 hour (in at 6:45PM)
8. Plate (75ul) at 7:45PM, placed in 37C incubator at 8:00PM

Extra cells were stored in the fridge.

Next: Mini-prep

September 5

Today: Seeding

Seeding of 20 colonies of our ligation from the plate. There were a lot of colonies, meaning that most of the vectors probably re-circularized without the insert.

Next: Mini-prep and screen

September 6

Today: Mini-prep of R+(B+C) transformed on Sept 4

We made 3 x 50ml batches of buffer P1 with RNAse added (5mg/50ml). They are in 50ml centrifuge tubes stored in the fridge.

1. Cultures removed from the SI at 1:15PM, and miniprep was done
2. Screening digest:
   1. We cut with BseRI (single cut in insert). We can tell by the length of the fragment if the ligation worked.

Conditions: 5ul DNA, 1.5ul buffer 2, 1.5ul BSA, 6ul water

1. 1. Because we used Elvis's method (no gel extraction), the insert could have simply re-inserted into its own vector instead of the R0062 vector. So we decided to use another enzyme to make sure we can see the presence of R0062.

September 7

Today: Decide on a new digest

We have some TaqI. If there is no R0062 (which is in the appropriate vector) there will only be 3 bands, if R was inserted there will be four bands. It's a bit of a long shot as it requires a complete digest or the gel will just be a mess of fragments (albeit an interpretable one).

Conditions: 5ul DNA, 1ul TaqI, 1.5ul buff 3, 1.5ul BSA, 6ul water, 65C, no star activity.

I used the heat block in the area where Raf usually is (to the left of our area). In the pink tray I did not find #14, 17, or 20, so I only ran 17 digests. I put the pink tray back in the -20C freezer. This is also where the Taq is. The digest was started at 5:50PM. Elvis will take it out when it finishes and put it in the freezer.

Next: Analyse the screen of the ligation.

September 10

Today: Mini-prep and screen

Today, a second attempt resulted in no indication that the ligation of B+C to R worked. We digesting the midiprep DNA with HindIII and XBal in the hopes to observe a small band of 100bp. After the digest we continued onto analyzing the digested DNA by running it for 25 minutes at 100 voltage on a 1.4% agarose gel. All 17 samples showed no bands at 100bp and only one bright band near the 2800-3000bp area, thus showing that at least DNA was present in our digests though not indicative as to whether or not the ligation worked due to the absent 100bp band.

In addition to that, we also did not see any DNA ladder working, and can therefore conclude that our positive control indicates either a failure in this DNA gel electrophoresis attempt or an erroneous 2-Log NEB ladder.

Next: Redo

September 12

Digest the B+C+R DNA with TaqI, and a gel was run. Analysis tomorrow.

September 13

Today: Analyse digest bands:

The gel seems to show only 3 bands, and some of them might have a fourth one. However its hard to distinguish things well because the ladders are really screwed up due to the Sybr Green.

Use 5ul per 50ml of TAE. So we used 10ul for 100ml of TAE.

September 17

Today: Overnight digest for yet another attempt at ligation

Overnight digest of the R0062 and the B0034+C0060 midipreps (2ul DNA each). That went into the incubation room at about 4:30PM.

Next: Check on digests tomorrow.

September 18

Today: Digest and Run gel with digested B+C
At 10:30AM, another 1ul of each enzyme was added to their respective digests. This is just to make sure that there are more cut DNA.

Gel was started at 3:50PM. The remaining 10ul of digested DNA was put into the tall -20C freezer on the top shelf.

Next: Check R and then ligate yet again.

September 24

Today: Transform R0062 Transformed the R0062 from the original plate, as well as the R0062 midiprep into top 10 cells. This was done in order to increase our pool of R0062, as we were running out of the midiprep. They were transformed and plated yesterday afternoon. We only had one plate left, so we plated half and half of the plate.

Next: Seeding

September 25

Today: Ran the old R on a gel to make sure that it was still okay

Next: Ligate.

September 26

Today: Extending the lives of incubating cells (aren't we nice)

Took half the seeding volumes and added additional LB and Amp. So we now have a total of 8 tubes (4 pairs). Shouldn't be a problem to miniprep since we can merge the pairs.

Next: Miniprep

September 27

Today: Miniprep

1. Standard mini-prep procedure. Screened and set up dilution for midiprep
2. Screening digest with TaqI: 1444bp, 445bp, 245bp
   1. Bad bands

Next: Midiprep

September 28

Today: Miniprep and ligation

Ligation Attempt #3728462 -- with Jay's help

1. Vector: 25ng/uL (~2000bp)
2. Insert: 5ng/uL (~800bp)
3. Want roughly 1:1 mass ratio, so 10ul insert + 2ul vector
4. Ligation conditions:
   1. 11ul insert
   2. 2ul vector
   3. 2ul ligase buffer
   4. 2ul T4 DNA ligase
   5. 3ul water
5. Vector-only control:
   1. 0ul insert
   2. 2ul vector
   3. 2ul ligase buffer
   4. 2ul T4 DNA ligase
   5. 14ul water

Ligation was left on desktop for over the weekend.

Next: Transformation

October 2007

October 1

Today: Transformation Transformed the ligation (R and B+C) and the control (R"cut" only) today into Top10 cells. Unfortunately, we forgot to use the high-efficiency cells. So if the ligation didn't work, we will have to transform again, but we still have quite a bit of the ligation left. The plates went into the incubator at around 9pm or so.

Next: Check on the plates tomorrow to see if there is growth.

October 2

Today: Seeding of the transformed (01/10/07) ligation (28/09/07)

1. Ten colonies from the 1X plate
2. Ten colonies from the 10X plate
3. Two controls: amp and no amp

Put into 37C SI at 4:05PM

Next: Mini-prep

October 3

Today: Mini-prep of ligation R+B+C

1. Standard mini-pep procedure. There is a bit more than 0.25ml left in some of the culture tubes because they had more LB/water put in than usual.
2. Contols were both clear when the cultures came out of the SI at 6:15PM.
3. Culture tubes are in the 4C in the lab.
4. DNA is in the large -20C freezer in the lab.

Next: Screen then midi-prep

October 5

Today: Analyzing the screen of the miniprep

1. It would appear that our ligation of R+(B+C) has worked, but the bands are not as nice as I'd like them to be. We did many digests for minipreps #10 and #12.
   1. EcoRI
   2. BsrGI
   3. PstI
   4. EcoRI + BsrGI
   5. EcoRI + PstI
   6. We also ran some of the un-digested miniprep DNA as control
2. The results were good, with an interesting twist. (By the way the pictures of the gel are saved as "Oct 5 ligation screen digest 2nd try")
   1. The single cuts all gave ~3000bp bands, which is great.
   2. The double cut with EcoRI + PstI showed a band at ~2100bp and ~800bp, which is good too.
      1. The double cut with EcoRI + BsrGI showed a band at ~2200bp (slightly higher than the 2100bp) and ~700bp (slightly lower than the 800bp), which is rather strange because we're supposed to get a band at ~2500bp and at ~450bp. I am sure that the insert is present, and the whole plasmid is at the appropriate length, because if the insert isn't there, any digests with BsrGI would not work at all (BsrGI cuts in the C0060 part of the insert).

1. Midiprep of #10 and #12 has been done.
   1. Quantification:
      1. #10: 180ng/ul
      2. #12: 225ng/ul

Next: Screen the midiprep and get ready for yet another ligation.

October 10

Today: Screen a little more thoroughly with the midiprep

1. Further screens (BseRI and XmnI) show that we do actually have the appropriate bands!
2. However, we will be sending this DNA out for sequencing just to make sure it is the correct DNA

October 13

1. Plan of ligation into J brick
2. J-brick: J40001 (2159bp)
3. Plasmid: pSB1A3 (~2.3kb)
   1. Total length: ~4.5kb
   2. We're planning to put the R+B+C to the back of the J-brick, but it doesn't really where we put it because both the aiiA and the J40001 has their own promoter.

1. Large scale digest:
   1. J40001:
      1. 8ul DNA
      2. 2ul PstI
      3. 1ul SpeI
      4. 2ul Buffer 2
      5. 2ul 10X BSA
      6. 4ul H2O
   2. R0062+B0034+C0060:
      1. 6ul DNA
      2. 1ul PstI
      3. 2ul XbaI
      4. 2ul Buffer 3
      5. 2ul 10x BSA
      6. 7ul H2O

1. Gel Extraction:
   1. 2 lanes of R+B+C (5ul digest each)
   2. The ~800bp bands were quite weak
   3. No DNA after gel extraction was done

October 14

After a whole weekend of trying to move our experiments along (with the J40001 (J brick) and the aiiA (R+B+C)), I can say that this whole gel extraction business is pretty strange. Allan did gel extraction of the R+B+C, and we came up with no DNA (ran on gel, and it showed absolutely nothing). This was done following the Qiagen protocols for gel extraction. Maybe Allan is doing something wrong, so Justin is going to try it tomorrow. The gel extraction kit worked for Elvis, so it definitely should work for us.

I have placed another overnight digest of J40001 (SpeI and PstI) and R+B+C (XbaI and PstI) this afternoon, and Justin is going to try to run them on a gel tomorrow (Monday), and extract the R+B+C from the gel.

Next: Retry extractions and set up ligation

October 15

Today: Gel extractions of the Aiia (R+B+C)

We got DNA out of it, but the concentration is uber uber low (probably less than 30ng/ul). We have 2 tubes of 50ul of extracted DNA. We will try to precipitate the DNA tomorrow and use a smaller volume of EB (30ul) to pool the DNA together. This will hopefully give us a more concentrated sample.

Next: Precipitate and get more concentrated sample.

October 16

Attempted to precipitate and concentrate the R+B+C gel extract:

1. Add 3 volumes of 100% isopropanol
2. Incubate in fridge (4C) for 12 hours
3. Centrifuge 10min at max speed
4. Decant supernatant
5. Add 300ul 70% ethanol
6. Centrifuge
7. Remove supernatant and let air dry
8. Add EB/TE

After this procedure, there was no DNA when run on a gel. So it was concluded that the procedure may have gone wrong and we lost the DNA. We need to re-do the gel extraction.

October 18

Today: Large scale digest of insert and vector and gel extraction

1. For both digests, one of the enzymes has 75% activity, so we put 2ul instead of 1ul.
   
   1. For J brick: 6ul DNA, 2ul PstI, 1ul SpeI, 2ul 10x BSA, 2ul Buffer and the rest with water to fill up to 20ul in total volume.
   2. For R+B+C: also digest 6ul DNA, 2ul XbaI, 1ul PstI, 2ul 10x BSA, 2ul buffer 3 then water to fill up to 20ul
   3. Both digests ran for 3 hours. R+B+C was run on the gel and excized with a razor (the digest went well, but not completely. There was not enough time to do the extraction, so the gel with the DNA is stored in the fridge and the Vector DNA (digested) is in the freezer.

1. Justin also performed double digests of J40001 later that day (PstI and SpeI)

Next: Gel extraction and ligation.

October 20

Today: Gel extraction
Gel extraction failed. The R+B+C was re-midiprepped and we will try again.

1. Confirmation of the various tubes of miniprepped J40001 DNA with gel:
   1. Lane 1: DNA Ladder
   2. Lane 2: J40001 sample 1
   3. Lane 3: J40001 sample 2
   4. Lane 4: J40001 sample 3
   5. Lane 5: J40001 sample 4
   6. Lane 6: J40001 sample 5
   7. Lane 7: J40001 sample 6
   8. Lane 8: J40001 sample 7
   9. Lane 9: DNA ladder

1. Gel results:
   1. Lane 1: DNA Ladder is good
   2. Lane 2: No DNA
   3. Lane 3: Multiple strange bands
   4. Lane 4: Band at ~4.5kb
   5. Lane 5: No DNA
   6. Lane 6: Multiple strange bands
   7. Lane 7: Multiple faint strange bands
   8. Lane 8: Multiple faint strange bands + strong ~4.5kb band
   9. Lane 9: DNA Ladder is good

Next: Gel extraction and ligation.

October 23

Today: Large scale digest and Gel extraction

1. We made the total digest volume 50ul this time, and digested 15ul of DNA for about 5 hours.
   1. 2ul XbaI
   2. 2ul PstI
   3. 5ul Buffer
   4. 5ul 10X BSA
   5. 15ul DNA
   6. 21ul water
2. Gel extraction:
   1. 4 lanes: 10ul of digest in each
   2. The gel extraction was successful!!

Next: Ligation.

October 24

Today: Ligation

1. Ligation:
   1. 14ul insert R+B+C
   2. 2ul vector J
   3. 2ul buffer
   4. 2ul ligase
2. Control (vector only):
   1. 14ul H2O
   2. 2ul vector
   3. 2ul buffer
   4. 2ul ligase

Next: Transform the ligation DNA on Thursday morning, and plate by 11AM. Then we can seed colonies at 11PM at night. Then on Friday morning around 11AM, we can do a miniprep. Miniprep will take about an hour, and then digest an hour, and then gel will run for like 45 minutes. So, hopefully we will have results by 3PM.

October 25

1. Transformed ligation DNA and the control DNA (3ul each) into NEB high efficiency cells, and plated by 12PM (noon)
   1. 50ul Plate
   2. 250ul Plate
2. However by about 7PM, there was no indication that there was any growth. So, we left it in the incubation room for next day.

October 26

1. There were some growth on all the plates
2. Seeded plates from the 250ul plate
3. We will miniprep and confirm the ligation over the weekend.