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