We filled 2 more molds, 10R and unknown. Our biggest problem so far is cracks and holes; its unknown the best way to do it, as the tape might be the reason for contamination. Temp: 29.5

No growth in colonies 3, 4, 7, and negative control for growth.
Ellen did plasmid minipreps of colonies 1, 2, 5, 6, 8 - 15.

Restriction Digest (12 plasmid preps total) with EcoR1 and Pst1 was run at 37*C at 9:45pm for 30 minutes.

Master Cocktail (13):
52ul H2O
13ul PstI
13ul EcoRI-HF
26ul 10x NEBuffer 2
26ul 10x BSA
TOTAL = 130ul

10ul of cocktail mix and 10ul of DNA were combined.

RE digest products were run on a 1% gel for 40 minutes to see if an insert of 4kb comes out of the 2kb pSB1C3:

20ul DNA digest product
2ul of 10x bromophenol blue + xylene cyanol dye.
Mixed and loaded 20ul in each lane.

Lane 1: 10ul 1kb PLUS ladder
Lane 2: 20ul of colony 1
Lane 3: 20ul of colony 2
Lane 4: 20ul of colony 5
Lane 5: 20ul of colony 6
Lane 6: 20ul of colony 8
Lane 7: 20ul of colony 9
Lane 8: 20ul of colony 10
Lane 9: 20ul of colony 11
Lane 10: 20ul of colony 12
Lane 11: 20ul of colony 13
Lane 12: 20ul of colony 14
Lane 13: 20ul of colony 15

Results:

Cannot see 4kb insert in any lanes, and can see 2kb backbone in lanes 13, 14, 15.

Both plates showed colony growth, with Gibson Construct plate #1 (higher dilution) showing more growth than Gibson Construct plate #2 (lower dilution).
Negative control (E. coli no plasmid) showed no growth.

Made Chlor LB broth media:
250ml LB
184ul stock Chloramphenicol per 50ml

Picked 15 colonies and grew overnight in 4ml culture tubes
Used older LB broth in fridge. Poured one negative control for growth.
All in incubator at 37*C overnight.

PCR Purification (A, N, U)
(Invitrogen)

· Add 4 volumes of PureLink Binding Buffer (B2) with isopropanol to 1 volume of the PCR product (50–100 μL). Mix well.
· Add the sample with the appropriate Binding Buffer (from step 1 of this procedure) to the PureLink Spin Column.
· Centrifuge the column at room temperature at 10,000 × g for 1 minute. (Discard the flow through)
· Add 650 μL of Wash Buffer with ethanol to the column.
· Centrifuge the column at room temperature at 10,000 × g for 1 minute. Discard the flow through from the collection tube and place the column into the tube.
· Centrifuge the column at maximum speed at room temperature for 2–3 minutes to remove any residual Wash Buffer. Discard the collection tube.
· Place the spin column in a clean 1.7-mL PureLink Elution Tube supplied with the kit.
· Add 50 μL of Elution Buffer
· Incubate the column at room temperature for 1 minute.
· Centrifuge the column at maximum speed for 2 minutes.
· The elution tube contains the purified PCR product. Remove and discard the column. The recovered elution volume is ~48 μL.

Made 1% Gel

· Ran in a gel for ~15-20 min

N U A(in plasmid) MWM

Cutting the gel and purifying

· Equilibrate a water bath 50 C
· Cut the gel
· 3 volumes (288ul)
· Place it in water bath for 10min
· Additional 5min

Purification procedure using centrifugation

· Load the dissolved gel mixture with DNA onto the center of a pure link wash tube
· Centrifuge the column at room temperature at 10,000 × g for 1 minute. (Discard the flow through)
· Add 650 μL of Wash Buffer with ethanol to the column.
· Centrifuge the column at room temperature at 10,000 × g for 1 minute. Discard the flow through from the collection tube and place the column into the tube.
· Centrifuge the column at maximum speed at room temperature for 2–3 minutes to remove any residual Wash Buffer. Discard the collection tube.
· Place the spin column in a clean 1.7-mL PureLink Elution Tube supplied with the kit.
· Add 50 μL of Elution Buffer
· Incubate the column at room temperature for 1 minute.
· Centrifuge the column at maximum speed for 2 minutes.
· The elution tube contains the purified PCR product. Remove and discard the column. The recovered elution volume is ~48 μL.

PCR
10ul plasmid prep a4
1ul 10X buffer
1ul Spe1

Made 1% Gel for PCR Purification for plasmid prep a4
(Also ran with Nag1 and Uap1)
First well DNA ladder (New England Bio quick-load), second well AGM1 plasmid,
Third well NAG1, and fourth well UAP1

Very little yield for linearized AGM1 plasmid but it is there. Std was 5uL, all others 10uL per lane. The smaller. The frag the more efficient the purification.

Transformation (Gibbson assembly)

Version 1 Version 2

7ul AGM1 plasmid 9ul AGM1 plasmid
1ul NAG1 0.5 NAG1
2ul UAP1 0.5 UAP1

10ul 10ul
+ 10ul master mix + 10ul master mix

20ul total 20ul total

*PCR (version 1 and version 2) is in PCR machine

Lab strain looks much better and uniform from last time, a sheet is expected. Ph 4. 5
Wild strain is not growing quickly but its growth seems more concentrated. Ph 4
Temp. 23.5 degrees.

We moved the molds out of the incubator and into cabinets in the lab. Four from before were contaminated, they were cleaned and four more molds were made. 5 star shapes were also put together.
Ten smaller beakers were also inoculated with the mycelia and were originally in the fan space (fan off).

Came into lab today, 7 out of 16 molds were contaminated. It seems like the tapes used to seal up cracks are the culprits as most of the mold growing seemed to originate at the tapes. There needs to be a better way to seal things up. what was done: we merely cleaned out the contaminated trays and are leaving the rest for tomorrow.

PCR products of the N and U gene plasmid digests were retrieved and run on a 1% gel at 3:08pm for about 40 minutes.

5ul of 1KB Plus DNA ladder

2uL PCR product
2ul Xylene Cyanol loading dye
10ul H2O
14ul total

Lane 1: empty
Lane 2: 1KB PLUS DNA ladder
Lane 3: Tube A (NAG1)
Lane 4: Tube B (NAG1) + MgCl
Lane 5: Tube C (UAP1)
Lane 6: Tube D (UAP1) + MgCl
(Recall that we did not have the proper reverse primer for AGM1, so this was not included)

Predicted band sizes:
Lane 3: 747kb
Lane 4: 747kb
Lane 5: 1461kb
Lane 6: 1461kb
(with lanes 4 and 6 showing more distinct bands, if the PCR reaction was enhanced by the MgCl cofactor)

Also, running the gel for 40 minutes was way too long-- next time only 25-30 so our product doesn’t go off the gel!

Results:

PCR products (tubes A, B, C, D) are in the hot pink “Cloning Materials” box and labeled “Genes N/U RE & PCR Products 8/23”

We ran a gel to confirm that we have PCR products of expected sizes. I glanced only quickly at the gel, saw that there were some bands (admittedly diffuse but the gel itself was a week old). The gel is attached (sad gel.png). Its name will become clear tomorrow.

The DNA sequences came back. None of the forward reactions worked (especially surprising as these primers were supplied by iGEM headquarters). Of the reverse reactions, some worked and some didn't. There was no read for the NAG1 gene, for example, but as we only had one digest that looked positive, we have to move forward with this clone (for now).

The best hits were:

AGM1 - clone 4
NAG1 - clone 5
UAP1 - clone 3

The next day we had lots of colonies (I don't have the pictures of the plates, but maybe someone else does). Many of these were red, indicating they were transformed due to residual RFP plasmid (as had happened previously). We circled several white colonies as prospective positive transformants. For each construct, we chose 5 clones and inoculated 4 ml of LB-chlor to grow these overnight at 37 deg C.

Gibson, Transformation:
So instead of 50 fentomoles of each, try 100 but 50 of the plasmid as before. I think we have room in the 10 microliters for that. And maybe close the lid of the PCR machine?

Gibson assembly protocol

-reaction is in 0.2mL PCR tube using program “Gibson” under user “ellen”
50C for 1h, hold at 4C
-total reaction volume is 20 microliters
-all G-Blocks were resuspended at a concentration of 50 fentomoles per microliter
-TOTAL amount of DNA in tube (sum of all parts) should be between 200-1000 fentomoles. NOTE: last time we were at the low end of this!

Recommended procedure:
For ‘U’
1.85 µL U-1
1.85 µL U-2
1.85 µL U-3
1.85 µL U-4
2.6 µL pSB1C3
10.0 µL Gibson Master Mix
20.0 µL

For ’N’
2.5 µL N-1
2.5 µL N-2
2.5 µL N-3
2.5 µL pSB1C3
10.0 µL Gibson Master Mix
20.0 µL

Results of Gibson Assembly:
4 big plates of the 3 chitin path genes A, U, and N, a + control
2 small plaets: neg control, baseline control without antibio

Results:

Lawn on antibiotic free control

Some contamination on A & U plates

No growth on N, + Control plates

[PASTE ALL PHOTOS FROM ALEX EMAIL AFTER RESIZING]

Pink colonies may be traces of RFP plasmids remaining from when biobrick plasmid part was PCR’ed.

Reasons for failure could be:

1) use the heated lid on the PCR machine - I did not shut it because I thought it might jack up the temp.
2) the manual says to use a 2-3x overage of inserts to backbone. The tech service guy said use equimolar so we did. maybe we need to rethink that.
3) overall total conc of DNA in the rxn mix could be upped.

G-blocks arrived