UCONN iGEM

Experiments

Describe the experiments, research and protocols you used in your iGEM project: protocols, experiments, designs

Protocols

FastCloning

Fastcloning of TrkH/TrkG into linearized pSB1C3

PCR Amplification

PRIMERS
SB-prep-3P-1
SB-prep-2Ea

	Reaction Setup:
	Reagent	[Final]	Volume (µL)	# of Rxns	Total Vol.
	ddH2O		17.5
	5x iProof buffer	1x	5
	10mM dNTP mix	0.2 mM/dNTP	0.5
	50pmol/µL For	0.5 µM	0.5
	50pmol/µL Rev	0.5 µM	0.5
	DNA Temp (50ng/µL)	1 ng/µL	0.5
	iProof DNA Pol (2U/µL)	0.04 U/µL	0.5
			25
		PCR CYCLE				Date:
			Temp(°C)	Time
	First Denature		98	3 mins		Start Time:
	Denature		98	30 secs		End Time:
	Anneal		65	30 secs	X 18 cycles
	Extension		72	2.5 min
	Last Extension		72	3 mins
			HOLD AT 4°C

Analysis of PCR products
1) Make 1% agarose gel (0.5 g agarose + 50 ml TAE) add 5µl of 10,000x Sybrsafe
2) Prepare samples: 2.5µL plasmid + 7.5 µL ddH2O +2µL 6xLB
Standard = 1µL standard (Invitrogen 1kb plus) + 9µL ddH2O+ 2µL 6xLB
3) Run until resolved starting at 80 Amps.

Dpn I Digestion of Parental Plasmid						Date:

Components	Init Conc	Fin.Conc.	Amount
ddH₂O	---	---	_______
DNA (total)	< 1µg	50 ng/µL	_______
10X CutSmart	10X	1X	2 µL
Dpn I	10 U/µL	0.5 U/µL	1 µL
		Total	20µL

1) Incubate on heat block at 37°C for 2 hours. ______-_______
2) Heat denature the Dpn I enzyme by incubating at 80°C for 20 mins. ______-______
3) Store at -20 °C or proceed directly to ligation reaction.

Transformation of Competent Cells with FastCloning Linearized Insert and Vector
1. Retrieve MC1061 cells from -20°C freezer. Thaw on ice started at ________ and quantity_______
2. Warm LB+Cam plates in the 37°C incubator. Started at ________
3. Pre-chill eppendorf 1.5ml tubes
4. Set up the following ratios of insert to vector and add 3 µL of each to a chilled eppendorf tube.
	3 insert :1 Vector
	2 Insert: 1 vector
	1 insert: 1 vector
6. Add 50 µL of MC1061 cells to each of the ___ tubes, mix gently.
7. Incubate on ice for 30 mins, gently flicking tube every 10 mins (_______ to _______)
8.Heat shock : Incubate at 42°C for 2 mins (_______ to _______)
9. Cold shock : Place cells on ice for 5 mins (_______ to _______)
10. Add 1 ml of SOC and place in shaking incubator (250 rpm) for >30 mins ( _______ to _______)
11. Spin down cells at 12k RPM for 60 secs, aspirate supernatent and resuspend in 100 µL LB.
12. Transfer the 100 µL in each tube to a labeled LB+Cam plate.
13. Place in incubator at 37°C ________
14. Removed at _________: continue to 5 ml cultures.

Selection and Amplification of Colonies
Preparation of 5 mL Cultures

1)	From LB stock aliquot 4 ml into sterile culture tubes.
2)	Add 4 µL of 35 mg/ml Chloramphenicol from freezer stock to each tube, including control
	for a final [35 µg/ml]
	Choose individual colonies and innoculate media, 1 colony/tube
			Colonies isolated from ______ plate
			Total cultured tubes ____ including a control
3)	Incubated in shaking incubator at 37°C (250 rpm) at ________
	Stopped at _______ for a total incubation time of _________
V. Miniprep of Plasmid DNA
Followed Qiagen miniprep handbook (pg 23-24)
1. Overnight cultures were aliquoted into 15 ml Falcon tubes.
2. Cells were centrifuged in the 15 ml tubes at 12k rpm for 1 minute. Aspirate supernatent.
3. Resuspended each pellet in 750 µl of buffer P1(resuspension buffer w/ Rnase added kept in cold
box).
4. Add 750 µL of buffer P2 (Lysis Buffer) and invert tubes 5 times to mix. 5 minute lysis (____-____)
5. Add 1050 µL of Buffer N3 (Neutralization Buffer) and invert tube 5x to mix
6. Centrifuge at 13K for 10 mins (______-______)
7. Transfer supernatent to new 15 mL falcon tube and centrifuge supernatent again at 13K RPM
for 10 mins
7. Load 1 ml of supernatent into labeled spin columns and microfuge for 60 secs, decant FT
8. Wash: Add 750 µL of Buffer PE (Wash buffer w/ EtOH)
	2X microfuge steps for 60 secs each time, followed by decant of FT
9. Elute each rxn in 30 µL of nuclease free ddh2o, incubate for 2 mins followed by centrifugation.
		Combine like samples

Concentrations based of UV Analysis

Sample	Concentration	A230	A260	A280	A260/A280

Gel Purification

IX - Purify by Gel/PCR Column

NOTE: This follows the protocol for the Promega SV Gel and PCR cleanup kit and centrifuge step.

1. Weigh gel slice.

2. Place a SV Minicolumn into a collection tube.

3. Add Membrane Binding Solution at a ratio of 10μl of solution per 10mg of agarose gel slice.

4. Vortex (or invert) the mixture and incubate at 50–65°C for 10 minutes or until the gel slice is completely dissolved.

NOTE: The maximum capacity of the column is 350mg of gel mass dissolved in 350μl of Membrane Binding Solution per column pass. For gel slices >350mg, continue to pass additional sample through the SV Minicolumn until the entire sample has been processed. The maximal amount of agarose that can be processed through a single column is approximately 3.5g (10 × 350mg) total. For DNA fragments > 5kb, mix by inversion rather than vortexing to avoid shearing DNA.

5. Transfer up to 350μL dissolved gel sample to column and incubate 3 min at room temperature.

6. Centrifuge 16,000g for 1 min and discard flowthrough.

7. Add 700μL Membrane Wash solution, centrifuge 16,000g for 1 min and discard flowthrough.

8. Perform second was with 500μL Membrane Wash solution, centrifuge 16,000g for 5 min and discard flowthrough.

9. Dry membrane by centrifuging 16,000g for 1 min.

10. Transfer column to new, sterile 1.5mL tube and add 23.5μL Nuclease-free water. Incubate at room temperature for 4 min.

11. Centrifuge at 16,000g for 1 min.

12. Determine DNA concentration by NanoDrop.

13. DNA can be stored at -20°C

Ligation

X – Ligation

NOTE: Ratios need to be calculated based on insert and vector lengths. For a 5kb vector and 1kb insert, 50ng of vector corresponds to 10ng of insert. For a 10:1 insert to vector ratio, the ligation would contain 50ng vector and 100ng insert. Determine the limiting component and scale reaction accordingly to use maximum volume of correct 10:1 ratio. Do not exceed a 50μL reaction volume.

1. Assemble ligation reaction containing appropriate insert/vector DNA and buffer.

2. Incubate at 45°C for 5 minutes to ensure nothing is pre-annealed.

3. Add 1μL T4 DNA Ligase

4. Incubate 16°C overnight.

5. Incubate reaction at RT for 30 minutes

6. Purify by column cleanup, eluting in 12μL nuclease-free H₂O.

Transformation

XI - Transformation

NOTE: Chemically competent cells are temperature sensitive, do not allow them to warm up, this will lower transformation efficiency.

1. Remove competent cells from -80°C freezer and thaw on wet ice (10-15 minutes).

2. Add entire miniprep DNA (pre-chilled) to 100μL of cells on ice. Stir briefly with a pipet tip – DO NOT pipet up and down to mix as this can heat up cells and introduce bubbles.

3. Incubate on ice for 30 minutes.

4. Heat-shock cells by placing tube in a 37°C heat block for 1 min.

5. Return cells to ice for 2 minutes.

6. Add 950μL of room temperature SOC to the cells in the culture tube.

7. Place tubes in a shaking incubator at 250 rpm for 1 hour at 37°C.

8. Centrifuge at 1,000g for 1 min, remove and discard 950μL of supernatant, resuspending cells gently in remaining SOC (~150μL).

9. Plate entire sample of transformed cells on LB agar plates with the appropriate antibiotic.

10. Incubate plates upside down overnight at 37°C.

SOC Recipe:

· 10g of Tryptone

· 5g Yeast Extract

· 10g NaCl

· 980mL of ddH₂O

· Adjust to pH 7.5 with NaOH and autoclave