Fastcloning of TrkH/TrkG into linearized pSB1C3 
PCR Amplification 
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    
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
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
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



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.



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

Add 20mL of 1M glucose (0.2μm filtered)