IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week3/Chemical and Light

Summary Powerpoint by Amy™

File:7-7monmtg slides (compatible).ppt

Ongo-ing "Ex-periments"

Transformation of Parts into S1

Transformation of Lac/Tet + GFP into S1

7/3: 25 mL culture of S1 was grown for electroporation and transformation.

Plasmid Name	uL DNA transformed	Plates grew?	Picked colonies?	Miniprepped?	Glycerol Stocks?
P58B	8uL	many small colonies, no GFP
P59B	8uL	5 medium-sized orangish-pinkish colonies, GFP +
P12 (from Christina)	5 uL
P27	2uL	9 small/medium orangish colonies, also many very small white-ish colonies
P28	2uL	many tiny colonies, no GFP
P29	5uL	many tiny colonies, no Venus fluor
P30	5uL	2 small pinkish colonies, YFP+
P31	8uL
P32	5uL	many (>50) medium orangish-pinkish colonies, no YFP

Kemikale 'n' Lyte

RE digests

Repeated digests of P1, P18, P26, P38, P39, P45, P56, P57

	P1B	P18	P26A	P38B	P39A	P45B	P56B	P57B
DNA	10 μL	20 μL	3 μL	5 μL	5 μL	3 μL	10 μL	10 μL
10X NEB Buffer	buffer 3, 2.5 μL	buffer 2, 2.5 μL	buffer 3, 2.5 μL	buffer 2, 2.5 μL	buffer 2, 2.5 μL	buffer 3, 2.5 μL	buffer 3, 2.5 μL	buffer 3, 2.5 μL
25X BSA	1 μL	1 μL	1 μL	1 μL	1 μL	1 μL	1 μL	1 μL
REs (1 μL each)	XP	SP	XP	SP	SP	XP	XP	XP
Water	9.5 μL	0 μL	16.5 μL	14.5 μL	14.5 μL	16.5 μL	9.5 μL	9.5 μL

07/08: Digested pCDF with XP:

• 12.5 μL water
• 1 μL 25X BSA
• 2.5 μL 10X NEB Buffer 3
• 7 μL DNA
• 1 μL of each RE (XbaI and PstI)

07/08: All of these digested plasmids were run on a 1% low melt gel.

MXH's gel extraction protocol:

• Cut out the bands
• Freeze at –20C for at least 20'
• Spin 13000 RPM 10’
• Use supernatant for ligation
• Then do gel extraction on rest of gel (QIAGEN protocol) and combine with above

Here is a ligation protocol (used by TA and MXH):

• 2 μL 5X Dilution Buffer
• 2 μL vector
• 6 μL insert
• 10 μL 2X Rapid Ligation Buffer
• 1 μL ligase
• vortex briefly
• incubate at RT for 10'
• use 5 μL to transform 50 μL cells

Transformations/Minipreps of Parts from Registries

07/07: We transformed P42, P11, P51, P52, P77, P78, P17, Q01121, BBa_J06911, BBa_J06912 from both the 2007 and 2008 registries (P11 was available only from the 2008 registry). We also made a positive transformation control using pUC19 DNA that came with the TOP10 cells. We made three negative plate controls using mock transformed bacteria on Amp, Kan, and Cm plates. We used E1.

FAILED TRANSFORMATIONS

2007 -- P51 (Kan), Q01121 (Kan), P17 (Kan), P52 (Kan), P78 (Kan), P77 (Kan)

2008 -- P52 (Kan), P51 (Kan), P78 (Kan), P77 (Kan), Q01121 (Kan), P17 (Kan), P11 (Cm)

The Cm and Kan mock transformations (negative controls) also failed to grow.

Alarmingly, the E1 mock transformation (no DNA, neg control) grew on the LB Carb plates. All of the Carb plates had some colonies, so we only picked from those with significantly higher numbers than the neg control. 3 colonies each from BBa_J06911 & BBa_J06912 2007 (temp sensitive LacI system) were picked and grown.

Successful Transformations

2007: P84, P85 (BBa_J06911, BBa_J06912) -- miniprepped/glycerol stocks made

The Amp negative control colonies did not grow in liquid LB Amp. The Amp positive control did grow in liquid LB Amp (pUC19).

Nanodrop of Minipreps

DNA concentrations

Part PCRs

07/07: More CDF was PCRed (w/ BioBrick adapting primers): 45μL PCR supermix, 1μL S1P13 (435ng/μL), 1μL CDF-F primer (20μM), 1μL CDF-R primer (20μM), 2μL water. Conditions: 5min @ 94°C → 35x[45s @ 94°C → 45s @ 58.7°C → 1m45s @72°C] → 5min @ 72°C → ∞ @ 4°C, heated lid

07/08: P11 and P17 from 2008 (left from punch), and P26, P38, P39, P42, P51, P52, P77, P78, Q01121 from 2007 plate were PCRed: 45μL PCR supermix, 1μL DNA, 1μL BBpfx primer (20μM), 1μL BBsfx (20μM), 2μL water. Conditions: 5min @ 94°C → 35x[45s @ 94°C → 45s @ 59°C → 1m45s @72°C] → 5min @ 72°C → ∞ @ 4°C, heated lid We're PCRing these b/c transformation for amplification has not been going smoothly.

Ligations

mtrA and p45

7/7/08 Ligated mtrA and p45 using standard protocol from ligation kit. In step 2 5uL of Vector DNA was added to make a 20uL dephosphorylation mix. In step 6, 4uL of the dephosphorylated Vector DNA and 4uL insert DNA were added.

Mutant Strand Syntheis Reaction

7/8/08 Used Stratagene QuikChange Site-Directed Mutatgenesis Kit (protocol) to create a point mutation in the pstI site in mtrA. Used 5ug of primer.

Gradient PCR

7/8/08 Used gradient PCR to PCR mtrB. Cycled 30-40°C x 10 and then 52-62°C x 30.

RE digests

We digested the following plasmids (per the Master Plan sent out to the iGEM google group):

• A (BBa_Q01121) with ES
• P11 with ES
• P17 with ES
• P23 with EX
• P24 with EX
• P26 with XP
• P38A with ES
• P38B with XP
• P39A with ES
• P39B with XP
• P42 with XP
• P56 with EX
• P58 with EX
• P59 with EX
• P63 with ES
• P77 with ES
• P78 with ES

These digests were run on gels and the bands of the correct size were extracted.

3% gel

• 100 bp ladder
• P38A with ES: 35 (NO BAND)
• P38B with XP: 35 (NO BAND)
• P39A with ES: 35 (NO BAND)
• P39B with XP: 35 (NO BAND)
• P63 with ES: 95 (extracted)

1.5% gel

• 100 bp ladder
• A (BBa_Q01121) with ES: 1372 (NO BAND)
• P17 with ES: 902 (NO BAND)
• P26 with XP: 961 (NO BAND)
• P42 with XP: 1128 (NO BAND)
• P77 with ES: 1307 (NO BAND)
• P78 with ES: 1310 (NO BAND)
• CDF with XP: ~900 (NO BAND)

1.5% gel

• P11 with ES: 4333 (NO BAND)
• P40 with SP: 2092 (extracted)
• P23 with EX: 2157 (extracted)
• P24 with EX: 2187 (extracted)
• P56 with EX: 2752 (extracted)
• P45A with EX: 2955 (NO BAND)
• P58 with EX: 3016 (extracted)
• P45A with EX: 2955 (NO BAND)
• P59 with EX: 3201 (extracted)
• 1 kb ladder