"

From 2013.igem.org

7/14/2013        Room temp for 1 hr

Ligation

I5-6-5 insert        i5-6-6 vector

<a href="#" name="d699bff16c8d8127072f23e97747d2e3e0204948"></a><a href="#" name="2"></a><tbody></tbody>

	3:1	0:1
DNA vet	2.6	2.6
Insert	6.25	0
Buffer	2	2
Ligase	1	1
Water	8	14.4

7/15/2013

Transformation Experimentation

Two types of cells

NEB 5-Alpha (High Efficiency)

• Heat shock 30s
• Ice for 5 mins
• 450 ul pre-warmed SOC
• 1hr incubation
• Plate 150 ul

Subcloning Efficiency (Invitrogen)

• Mix cells gently before remaining from tube
• Don’t mix DNA into cells by pipetting, tap gently
• Heat shock 20s
• Ice for 2 mins
• 450 ul SOC
• 1 hr incubation
• Plate 150 ul

[pLac]        i5-6-3         NEB: i5-7-1        invitrogen: i5-7-5

[RFP] Kit plate 3. Well 12 W. NEB: i5-7-2. invitrogen: i5-7-6

[wintergreen] kit plate 3. Well 5E. NEB: i5-7-3. Invitrogen: i5-7-7

I4.47.5                                Neb: i5-7-4. Invitrogen:i5-7-8

7/16/2013

Transformation Results

<a href="#" name="9fa66a2272f48af476915e7061cb17b2434d7747"></a><a href="#" name="3"></a><tbody></tbody>

	# of colonies	# of colonies
	NEB	Invitrogen
[pLac] i5-6-3	50+	0
[RFP] for plate	30+	4
[wintergreen] from plate	22	2
I4.47.5	TMIC at least 100+	10+

[RFP] from plate (i5-7-2) →  30+ colonies →  picked 2 colonies → i5-8-3

                                                                        →  i5-8-4

Ligation

Insert: i5-6-5        Vector: i5-6-6

<a href="#" name="44cca1f0fd090f0eb31f993d7b54030e64a4847d"></a><a href="#" name="4"></a><tbody></tbody>

	3:1	0:1
Vector	2.6	2.6
Insert	6.25	0
Buffer	2	2
Ligase	1	1
Water	8	14.4
Total	20	20

Room temperature for 1 hr

Transformation

→ NEB5X cells; 30 secs heat shock

3:1 →  i5-8-1

0:1 → i5-8-2

7/17/2013

Miniprep

I5-8-3        120.5 ng/ul        [RFP]

I5-8-4        160.0 ng/ul        [RFP]

Transformation

I5-6-3 (positive control) →  i5-9-1 [pLac] →  0 colonies

3:1                        → i5-9-2 [pLac-GFP] → 0 colonies

0:1                        → i5-9-3 [pLac vector] → 3 colonies

7/19/2013

Inoculation from glycerol stock:

[Lac I-RBS-RFP] – I3-5-10

[Promoter-RBS-GFP] – I3-7-7 and I3-7-8

Population 1: I3-60-4

Population 2: I3-46-6

Population 3: I3-46-4

Transformation

Positive Control

[pTet] Kit plate 5-2013. Well 6I. Resistance chlora. I5-9-4.

[RBS] Kit plate 5-2013. Well 2M. Resistance Amp. I5-9-5.

[Constitutive Promoter] Kit plate 5-2013. Well 18C. Resistance Amp. I5-9-6.

7/20/2013

0 colonies on all transformations