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Protocols

Biolab Protocols

Agarose gel

AG Ignatova

  • 0.5 g Agarose
  • 50 mL TAE-buffer (gel room)
  1. Heat in microwave until agarose is dissolved
  2. Transfer into gel slide, tip pipet into EtBr, then into gel
  3. Cool for 30 minutes

Annealing

AG Ignatova

  1. get the amount of DNA from the delivery note
  2. dilute both oligos to 100 μM
  3. anneal in water bath at 94 °C for 10 min
  4. cool to room temperature in water bath over 60 min
  5. store solution at 4 °C in fridge

Annealing Buffer

AG Ignatova

  • 0.5 M EDTA (provided by Dr. Shinichiro Komaki)
  • 5 M NaCl (provided by Dr. Shinichiro Komaki)
  • 1 M Tris (self made: made in 10 mL ddH2O, pH 8.04)

for 10 mL:

EDTA 20 μL
Tris 100 μL
NaCl 100 μL
+ H2O → 10 mL

Backbone dephosphorylation

AG Ignatova

10 μL pSB1C3 (cut)
1 μL FastAP
4 μL FastAP Buffer

→ 37 °C for 30 minutes

→ purify with PCR clean up kit

Cell culture b How to passage and split cells (HeLa cells)

Frauke Adamla

Reagents needed (for HeLa cells):

  • 1x PBS
  • DMEM media
  • 0,05% Trypsin/EDTA
  • Gentamycin (G418; 50 mg/mL in PBS sterile filtered)
  • Hygromycin B (Life Technologies; 50 mg/mL)
  • In case of stopping expression (TetOff-System): Doxycyclin

General hints:

  • Wear gloves
  • Disinfect yourself as well as the clean bench before using with 70% EtOH
  • Everything else that is put under the clean bench is disinfected (bottles, pipettes, racks etc.)
  • Cell bottles as well as dishes are touched only with gloves
  • Try to keep cells in 37 °C incubator as long as possible (so they are the last thing you fetch when you begin to work; avoid keeping them at RT)
  • Media, PBS-buffer and trypsin are prewarmed at 36 °C in water bath (for at least 20-30 min; avoid keeping solutions at 36 °C the entire day)
  • Clean the bench after using; charge pipette boy and remove all your stuff you have used

How to passage and split cells (T-75 bottle):

  1. Check confluence of cells with microscope: cells should be 80-90% confluent when passaging, should build monolayer, should not grow on top of each other or build clumps, dead cells swim above
  2. Exhaust media with 1 mL disposable pipette (break filter) in corner of the bottle to avoid contact with cells
  3. Add 3 mL PBS and wave to wash cells
  4. Exhaust buffer again with 1 mL pipette
  5. Add 3 mL 0,05% Trypsin (this time is can be pipetted on the cell lawn directly)
  6. Wave a bit and then incubate at 37 °C for ca. 5 min (you can also tap against bottle to dissolve the cells; check with microscope)
    → Meanwhile prepare media in falcon and add AB etc.
    → Sign 15 ml falcons for resuspended cells or new bottles/dishes
  7. Add 3 mL media; wash cells off and split cells by pipetting up and down (you do not have to be careful with HeLa cells; they are very tough; the best bet is to put pipette directly on the ground of bottle); avoid producing foam
  8. Transfer cells to falcon; try to remove foam
  9. Add media to bottles and then add cells; wave very good to distribute the cells (check with microscope)
  10. Sign the bottles (renew the passage and the date if it is an old bottle)
  11. Put cells at 37 °C
  12. Clean the bench

PBS Trypsin Media Media total Comparable to dish
6 well 2-3 mL 0,5 mL 0,5 mL 2,0 mL --
T25(= 25 cm2) 3,0 mL 1,5 mL 1,5 mL 5,0 mL 60 mm
T75(= 75 cm2) 5-6 mL 3,0 mL 3,0 mL 10-15 mL 100 mm
T175(= 175 cm2) 10 mL 5,0 mL 5,0 mL 25 mL 145 mm

Cell culture b Transfection of HEK cells

Frauke Adamla (according to Ingrid)

Passage and Enumerate HEK Cells

  • Aspirate the medium
  • Wash with PBS once (8-10 min for 10 cm dish)
  • Trypsinization: 3 mL pro 10 cm
  • Incubate for 3 min at 37 °C
  • Meanwhile, prepare new plates (label, distribute medium):
Dish size Volume Growth surface
6 cm 3 mL 20 cm2
10 cm 9 mL 60 cm2
3.5 cm (1 well) 2 mL 9.6 cm2 (10 cm2)
  • Don't use medium with AB for transfection, because the transfection-medium also channels AB into the cells, which is not good
  • Add 5 mL medium to the trypsinated cells
  • Transfer into a falcon and centrifuge: 3 min, 1200 rpm, RT
  • Discard the supernatant
  • Resuspend the pelleted cells in 10 mL
  • Pipette up and down at least 10x
  • Use 10 µL to count the cells (Ingrid: don't use Trypanblue, Neubauer-counting chamber)
  • Count 4 large squares: here e.g. 170 cells per square x 10= cells/µL; here 1700 cells/µL
  • Seed cells for transfection: 300.000 cells per 1 well (use Andreas) or 500.000 cells per 1 well(uns Ingrid)
  • 1.2 Mio cells for 6 cm for transfection, so 705 µL
  • For normal splitting: 1:40 (250 µL) HEK, when only split once a week

Transfection

  • Prepare the plasmids in Eppendorf tubes: 4.5 µg plasmid-DNA for 6 cm dish
  • Here: 4 µL plasmid, 14 µL PEI (store at 4 °C), fill up to 400 µL with Optimen
  • Centrifuge shortly
  • Set aside at RT for 30 min (under the hood or in the Thermoblock)
  • 6 well: growth surface= 9.6 cm2, 1.2 Mio cells
    → 6 cm dish: 21 cm2= 500.000 cells
  • Transfection: 6 well= 2 µg DNA, Fill up 7 µL PEI to 200 μL with Optimen; 6 cm= 4.5 µg DNA, 14 µL PEI @ 400 µL Optimum
  • Fill up to a total volume of 4 mL with DMEM
  • If AB was used, split the cells in medium without AB and seed cells out one day before
  • Aspirate the medium
  • Add 3.6 mL new medium
  • Slowly drizzle the transfection mix on it (then the total volume is 4 mL)
  • Traverse once to mix it
  • Put it in the incubator
  • Aspirate the medium after 4-6 hours and add new medium
  • After 24 hours the cells can be microscoped etc.

Chloramphenicol Stock

AG Ignatova

P4 mg/mL preparation of Choramphenicol Stock (3 in EtOH)

  1. dissolve 34 mg of Chloramphenicol in 1 mL 100% ethanol
  2. filter through a 0.22 μL filter to sterilize
  3. use at 1:1000 dilution in LB or LB-agar
  4. comment: prepare bigger stock next time (10 mL). It's not very exact working with 34 mg CAmp
  5. mark as CAmp (top) and CAmp/dd.mm.yy/34 mg/mL (side) in Antibiotics
  6. store at -20 °C

Competent Cells

AG Ignatova

Reagents

  • Cells
  • LB plate
  • LB medium
  • TB-PIPES buffer
  • Liquid nitrogen

Day 1

  1. Flame the metal inoculating loop
  2. Scrape off a portion from the top of the frozen (DH5-alpha) cells
  3. Streak it onto the LB plate
  4. Put the stock back to -80 °C immediately
  5. Leave the plates for 5 minutes and place them upside down in the 37 °C incubator overnight

Day 2

  1. Pick a single colony into 3 mL of LB medium
  2. Inoculate the culture over night at 37 °C with shaking at 200 rpm
  3. Transfer 0.5 mL of the overnight preculture into 50 mL LB medium (1-2 L Erlenmeyer flask)
  4. Let the culture grow to OD600 = 0.6 at 18 °C with shaking at 200 rpm
  5. Transfer the culture into a 50 mL Falcon tube and leave for 10 min on ice
  6. Spin down cells at 3500 rpm for 8 min at 4 °C
  7. Resuspend in 1/3 volume TB-PIPES buffer (17 mL for 50 mL original culture)
  8. Leave for 10 min on ice
  9. Spin down cells at 3500 rpm for 8 min at 4 °C
  10. Resuspend in 1/12.5 volume (4 mL for 50 mL original culture)
  11. Add 7% DMSO (sterile)
  12. Leave for 10 min on ice
  13. Aliquot 50 μL in pre-chilled Eppendorf tubes and freeze by immersion in liquid nitrogen
  14. Store at b��80 °C

Competent Cells

Zhang Gong

Buffers and solutions:

  • Ca/glycerol buffer: 60 mM CaCl2, 10 mM PIPES, 150 mL glycerol, Fill water up to 1 L, pH=7.0
    Filter sterilization. Avoid autoclaving.

Protocol:
  1. Grow the Cells at 37 °C till OD=0.2-0.4
  2. Chill the culture on ice for 5 min.
  3. Collect cells by centrifugation, 6000 rpm 10 min 4 °C
  4. Gently resuspend the cells from 500 mL LB in 40 mL cold Ca/glycerol buffer on ice.
  5. Incubate the cells on ice for 5˜30 min.
  6. Repeat step 3 and 4.
  7. Incubate the resuspended cells in Ca/glycerol buffer on ice for 30 min. (The longer, the better)
  8. Collect cells by centrifugation, 6000 rpm 10 min 4 °C
  9. Resuspend cells in 6 mL Ca/glycerol buffer.
  10. Aliquote 150 µL/tube. Freeze in liquid N2 and store at -80 °C.

Control of Annealing

AG Ignatova

  • in agarose gel (1%)
  • 1 μL loading dye
  • 9 μL sample (or appropriate amount & add H20 up to 9 μL)

DNA dephosphorylation and ligation

Frauke Adamla, AG Ignatova, University of Hamburg

  1. DNA dephosphorylation was necessary to avoid recircularization of plasmid vectors digested with restriction enzymes.
  2. For dephoshorylation, 1 μL CIP was added together with 1x NEBuffer 3 to the sample and incubated at 37 °C for 20 min.
  3. Following dephosphorylation, linearized plasmid DNA was purified with GeneJET PCR purification Kit and used for DNA ligation with an insert obtained from PCR or DNA restriction digest using T4 DNA ligase.
  4. Ligation was carried out in 20 μL sample volume containing 1x DNA ligase buffer, 1 μL T4 DNA ligase and 40 ng vector DNA at different molar ratios of insert (e.g. 1:4, 1:8).
  5. Afterwards, samples were incubated at 22 °C for 60 min. Subsequently, the ligation mixture was used for transformation into chemically competent E. coli DH5α.

GeneJET Gel Extraction Kit

#K0691, #K0692

Thermo SCIENTIFIC Product Information

PURIFICATION PROTOCOLS

Note

  • Read IMPORTANT NOTES on p.3 before starting.
  • All purification steps should be carried out at room temperature.
  • All centrifugations should be carried out in a table-top microcentrifuge at >12000 x g (10000-14000 rpm, depending on the rotor type).

Protoco  A. DNA extraction from the gel using centrifuges

Step Procedure
1 Excise gel slice containing the DNA fragment using a clean scalpel or razorblade. Cut as close to the DNA as possible to minimize the gel volume. Place the gel slice into a pre-weighed 1.5 mL tube and weigh. Record the weight of the gel slice.
Note. If the purified fragment will be used for cloning reactions, avoid damaging the DNA through UV light exposure. Minimize UV exposure to a few seconds or keep the gel slice on a glass or plastic plate during UV illumination.
2 Add 1:1 volume of Binding Buffer to the gel slice (volume: weight) (e.g., add 100 μL of Binding Buffer for every 100 mg of agarose gel).
Note. For gels with an agarose content greater than 2%, add 2:1 volumes of Binding Buffer to the gel slice.
3 Incubate the gel mixture at 50-60 °C for 10 min or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to facilitate the melting process. Ensure that the gel is completely dissolved. Vortex the gel mixture briefly before loading on the column.
Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 μL of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.
4
for ≤ 500 bp and > 10 kB DNA fragments 		Optional: use this step only when DNA fragment is ≤ 500 bp or > 10 kB long.
  • If the DNA fragment is ≤ 500 bp, add 1 gel volume of 100% isopropanol to the solubilized gel solution (e.g. 100 μL of isopropanol should be added to 100 mg gel slice solubilized in 100 μL of Binding Buffer). Mix thoroughly.
  • If the DNA fragment is >10 kb, add 1 gel volume of water to the solubilized gel solution (e.g. 100 μL of water should be added to 100 mg gel slice solubilized in 100 μL of Binding Buffer). Mix thoroughly.
5 Transfer up to 800 μL of the solubilized gel solution (from step 3 or 4) to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
Note.
  • If the total volume exceeds 800 μL, the solution can be added to the column instages. After each application, centrifuge the column for 30-60 s and discard the flow-through after each spin. Repeat until the entire volume has been applied to the column membrane. Do not exceed 1 g of total agarose gel per column.
  • Close the bag with GeneJET Purification Columns tightly after each use!
6 Optional: use this additional binding step only if the purified DNA will be used for sequencing.
Add 100 μL of Binding Buffer to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
7 Add 700 μL of Wash Buffer (diluted with ethanol as described on p.3) to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
8 Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove residual wash buffer.
Note. This step is essential to avoid residual ethanol in the purified DNA solution. The presence of ethanol in the DNA sample may inhibit downstream enzymatic reactions.
9 Transfer the GeneJET purification column into a clean 1.5 mL microcentrifuge tube (not included). Add 50 μL of Elution Buffer to the center of the purification column membrane. Centrifuge for 1 min.
Note.
  • For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 μL does not significantly reduce the DNA yield. However, elution volumes less than 10 μL are not recommended.
  • If DNA fragment is >10 kb, prewarm Elution Buffer to 65 °C before applying to column.
  • If the elution volume is 10 μL and DNA amount is ≤ 5 μg, incubate column for 1 min at room temperature before centrifugation.
10 Discard the GeneJET purification column and store the purified DNA at -20 °C.

GeneJET PCR Purification Kit

#K0701, #K0702

Thermo SCIENTIFIC Product Information

PURIFICATION PROTOCOLS

Note

  • Read IMPORTANT NOTES on p.3 before starting.
  • All purification steps ahould be carried out at room temperature.
  • All centrifugations should be carried out in a table-top microcentrifuge at >12000 x g (10000-14000 rpm, depending on the rotor type).

Protocol A. DNA purification using centrifuge

Step Procedure
1 Add a 1:1 volume of Binding Buffer to completed PCR mixture (e.g. for every 100 µL of Binding Buffer). Mix thoroughly. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 µL of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.
2
for DNA ≥500 bp 		Optional: if the DNA fragment is ≥ 500 bp, add a 1:2 volume of 100% isopropanol (e.g., 100 µL of isopropanol should be added to 100 µL of PCR mixture combined with 100 µL of Binding Buffer). Mix thoroughly.
Note. If PCR mixture contains primer-dimers, purification without isopropanol is recommended. However, the yield of the target DNA fragment will be lower.
3 Transfer up to 800 µL of the solution from step 1 (or optional step 2) to the GeneJET purification column. Centrifuge for 30-60 s. Discard the flow-through.
Note. If the total volume exceeds 800 µL, the solution can be added to the column in stages. After the addition of 800 µL of solution, centrifuge the column for 30-60 s and discard flow-through. Repeat until the entire solution has been added to the column membrane.
Close the bag with GeneJET Purification Columns tightly after each use!
4 Add a 700 µL of Wash Buffer (diluted with the ethanol as described on p.3) to the GeneJET purification column. Centrifuge for 30-60 s.
Discard the flow-through and place the purification column back into the collection tube.
5 Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove any residual wash buffer.
Note. This step is essential as the presence of residual ethanol in the DNA sample may inhibit subsequent reactions.
6 Transfer the GeneJET purification column to a clean 1.5 mL microcentrifuge tube (not included).
Add 50 µL of Elution Buffer to the center of the GeneJET purification column membrane and centrifuge for 1 min.
Note.
  • For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 µL does not significantly reduce the DNA yield. However, elution volumes less than 10 µL are not recommended.
  • If DNA fragment is > 10 kb, prewarm Elution Buffer to 65 °C before applying to column.
  • If the elution volume is 10 µL and DNA amount is ≥5 µg, incubate column for 1 min at room temperature before centrifugation.
7 Discard the GeneJET purification column and store the purified DNA at -20 °C.

GeneJET Plasmid Miniprep Kit

#K0502, #K0503

Thermo SCIENTIFIC Product Information

Growth of Bacterial Cultures

  • Pick a single colony from a freshly streaked selective plate to inoculate 1-5 mL of LB medium supplemented with the appropriate selection antibiotic. Incubate for 12-16 hours at 37 °C while shaking at 200-250 rpm. Use a tube or flask with a volume of least 4 times the culture volume.
  • Havest the bacterial culture by centrifugation at 8000 rpm (6800 x g) in a microcentrifuge for 2 min at room temperature. Decant the supernetant and remove all remaining medium.
Do not overload the column:

For high-copy-number plasmids (see Table 1), do not process more than 5 mL of bacterial culture. If more than 5 mL of such a culture are processed, the GeneJET spin column capacity (20 μL of dsDNA) will be exceeded and no increase in plasmid yield will be obtained.
For low-copy-number plasmids (see Table 1), it may be necessary to process larger volumes of bacterial culture (up to 10 mL) to recover a sufficient quantity of DNA.

Table 1. Copy numbers of various vectors

High-copy
300-700 copies per cell 		Low-copy
10-50 copies per cell 		Very low-copy
up to 5 copies per cell
pUC vectors
pBluescript vectors
pGEM vectors
pTZ vectors
pJET vectors 		pBR322 and derivatives
pACYC and derivatives 		pSC101 and derivatives

PURIFICATION PROTOCOLS

Note

  • Read IMPORTANT NOTES on p.3 before starting.
  • All purification steps should be carried out at room temperature.
  • All centrifugations should be carried out in a table-top microcentrifuge at >12000 x g (10000-14000 rpm, depending on the rotor type).

Use 1-5 mL of E. coli culture in LB media for purification of higt-copy plasmids.
For low-copy plasmids use up to 10 mL of culture.

Protocol A. Plasmid DNA purification using centrifuges

Step Procedure
1 Resuspend the pelleted cells in 250 μL of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
Note. Ensure RNase A has been added to the Resuspension Solution (as describe on p.3)
2 Add 250 μL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear.
Note. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.
3 Add 350 μL of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times.
Note. It is important to mix thoroughly and gently after the addition of the Neutralization Solution to avoid localized precipitation of bacterial cell debris.
The neutralized bacterial lysate should become cloudy.
4 Centrifuge for 5 min to pellet cell debris and chromosomal DNA.
5 Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white prepipitate.
Note. Close the bag with GeneJET Spin Columns tightly after each use!
6 Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
Note. Do not add bleach to the flow-through, see p.8 for Safety Information.
7
for EndA+ strains only 		Optional: use this preliminary washing step only if EndA+ strains which have high level of nuclease activity are used.
Wash the GeneJET spin column by adding 500 μL of Wash Solution I (#R1611, diluted)
8 Add 500 μL of Wash Solution (diluted with ethanol prior to first use as described on p.3) to the GeneJET spin column. Centriifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.
9 Repeat the wash procedure (step 8) using 500 μL of the Wash Solution.
10 Discard the flow-trough and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.
11 Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube (not included). Add 50 μL of the Elution Buffer to the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min.
Note. An additional elution step (optional) with Elution Buffer or water will recover residual DNA from the membrane and increase the overall yield by 10-20%
For elution of plasmids or cosmids >20 kb, prewarm Elution Buffer to 70 °C before applying to silica membrane.
12 Discard the column and store the purified plasmid DNA at -20 °C.

General transfection Protcol
Cell type used in this setup: HeLa

Adapted from Robert Rauscher

Day 1
  • Split cells into 3.5 cm (diameter) dish. 400,000 without antibiotics)
  • Pellet cells after trypsinization to remove trypsin
Day 2
  • Prepare transfection mixture using PEI40
  • 25 μL PEI40 + x DNA (2 μg DNA) + x μL OPTIMEM = 100 μL total
  • Incubate mixture at 22 °C for 20 min.
  • Aspirate old medium from cells and add 0.9 mL fresh medium
    • test
  • In a dropwise manner, add mixture to well (total volume is now 1 mL)
  • Incubate transfection reaction for 5 hours in 37 °C incubator, supplemented with 5% CO2.
  • Aspirate medium and wash with 2 mL PBS.
  • Add 2 mL of fresh medium

Illustration 1: Original taken from Roberts Lab Book

Glycerol stocks

AG Ignatova

from liquid culture

  • 500 μL (50% Glycerol, 50% H2O)
  • 500 μL BW29655
  • -80 °C

LB-Agar Plates (+ Chloramphenicol/ + Kanamycin)

AG Ignatova

  1. Heat 250 mL LB-Agar
  2. Cool down to ca. 50°C
  3. Add 250 μL Chloramphenicol/ 12.5 μL Kanamycin (50 μg/mL)/ 25μL Ampicillin (80 μg/mL)
  4. Preparation of 20 plates under sterile conditions
  5. Store at 4 °C, mark as LB CAmp/Kan/Amp Plates iGEM, dd.mm.yyyy

Ligation

AG Ignatova

1 μL pSB-1C3 vector (23 ng)
1 μL/1.5 μL GroEl-Insert (3.5 ng) / miRNA-Insert (3.3 ng)
2 μL T4 Buffer
1 μL T4 Ligase
5 μL ddH20 (6 μL for control)

15 min at room temperature, then 10 min at 65 °C

Lysis of cells (in 12-well or 6-well plates)

Justyna Hinz

  1. wash cells once with 1 mL of PBS
  2. trypsinize with 0.5 mL trypsin for 1 min at RT, add 0.5 mL medium and centrifuge at 2000 rpm for 5 min.
  3. carefully remove the supernatant, resuspend the cells in 100 μL lysis buffer, incubate with Benzonase (20 U/500.000 cells) for 45 min. -1 h on ice.
    When youB4re preparing the whole lysates they can be frozen and stored in -80 °C at this point. For separating the soluble and SDS-non-soluble protein fraction an solubilizing the latter one the lysates must be further processed:
  4. Spin down at 13000 rpm for 10 mins.
  5. Separate supernatant b

Media

Tejas

Content

LB-Medium
M9Y Medium
M9 minimal medium

LB Medium

Yeast extrakt 5 g/L
Trypton 10 g/L
NaCl 10 g/L
RO-water add 1000 mL
pH 7.5

autoclave: 20 min 121 °C


M9Y Medium (with yeast extract)

Na2HPO4 6 g/L
KH2PO4 3 g/L
NaCl 0.5 g/L
NH4Cl 1 g/L
Yeast extract 2.5 g/L
RO-water ad 1000 mL
pH 7.4
autoclave: 20 min 121 °C

After autocalving add following substances:
1 M MgSO4 2 mL
20% Glucose 15 mL (end conc. 3 g/L)
0.1 M CaCl2 1 mL

Glucose and MgSO4 autoclave separately, CaCl2 to be steril filtered.


M9 minimal medium

M9 salts (part 1) 10x:
Na2HPO4 68 g
KH2PO4 30 g
NaCl 5 g (do not add for osmotic stress experiment)
NH4Cl 10 g
Add water to 1 L. Adjust pH=7.4. Autoclave
M9 (part 2) 10x:
MgSO4 10 mM
Glucose 30 g
Add water to 1 L. Filter sterilization.

CaCl2 100 mM stock solution. Filter sterilization

AA-Met mixture 250 mg/L each, pH=7.4. Filter sterilization


To use:

10x M9 salts (part 1) 1 mL
10x M9 (part 2) 1 mL
5x AA 2 mL for stress experiment don't add!
CaCl2 100 mM 0.1 mL
Water up to 10 mL

PCR protocols

Zhang Gong

Content

  • PCR protocols
  • Fragment amplification with Herculase
  • Fragment amplification with Pfu
  • mutagenesis

Fragment amplification with Herculase

Tamplate 1 μL
Primers (100˜200 μM) 1 μL each
10x Herc buffer 5 μL
Water 39.5 μL
dNTPs (10 mM each) 1.5 μL
Herculase 0.5˜1 μL
50 μL
95 °C 2 min
95 °C 30 sec 10 cycles
55 °C 45˜60 sec
72 °C 1 min/kb
95 °C 30 sec 20 cycles
55 °C 45˜60 sec
72 °C 1 min/kb + 10 sec/cycle
4 °C -

Fragment amplification with Pfu

Tamplate 1 μL
Primers (100˜200 μM) 1 μL each
10x Pfu buffer 5 μL
Water 39.5 μL
dNTPs (10 mM each) 1.5 μL
Pfu polymerase 1 μL
50 μL
95 °C 2 min
95 °C 30 sec 30 cycles
55 °C 45˜60 sec
72 °C 2 min/kb + 1 min
4 °C -

Mutagenesis

Tamplate (1:5 dilution) 1 μL
Primers (10 μM) 1 μL each
10x Pfu buffer 5 μL
Water 39.5 μL
dNTPs (10 mM each) 1.5 μL
Pfu polymerase 1 μL
50 μL
95 °C 2 min
95 °C 30 sec 18 cycles
55 °C 45˜60 sec
68 °C 2 min/kb + 1 min
4 °C -
DpnI 1 μl, 37 °C 1 h. Then transform 5 μL into DH5α.

Note:

  1. If you get too less colonies, you may think to increase the cycles to 20, but no more! Otherwise you might get mistakes in the final sequence.
  2. You can try both Pfu and Herculase to perform muatgenesis. Pfu will give less yield but more fidelity, so you should preferably use Pfu. If you cannot succeed with Pfu then you can consider trying Herculase. Just keep the same protocol!

Pick colony

AG Ignatova

  • Inoculate each picked colony into 5 mL of liquid LB-(CAmp/ respective antibioticum)-medium, respectively
  • Tubes in the 37 °C incubation room, on rotator!

Preparation for sequencing

AG Ignatova

  • Primer concentration: 100 μM → Concentration needed: 5 μM (dilute at 1:20)
  • take 50 nm of sample mix with 5 μL of (5 μM primer, only forward)

Restriction

AG Ignatova

20 μL pSB-1C3 (500 ng)
1 μL EcoRI
1 μL PstI
3 μL FD Buffer
5 μL ddH2O

20 minutes at 37 °C, following clean up with kit

SOC medium

Dagmar Stang

Bacto-Tryptone 2.000g 
Bacto-Hefe-Extract 0.500 g
NaCl 0.050 g
KCl 0.019 g
MgCl2 * (H2O)6 0.203 g (without (H2O)6 → 0.095 g)
Glucose 0.036 g
pH 7.0 with pH-Meter
→ 10 mL (fridge)

TB PIPES Buffer

AG Ignatova

for 100 mL:

KCl 1.865 g
CaCl x 2 H2O 0.220 g
0.5 M PIPES 2 mL
pH 6.7
MgCl2 x 2 H20 0.889 g
ddH2O → 100 mL

Transformation

AG Ignatova

10 μL ligation product
50 μL competent cells
  • 30 min on ice
  • 1 min Heatshock at 42 °C
  • add 200 μL SOC-Medium
  • 2 h at 37 °C on rotator
  • plate out on appropriate plates (C-Amp/ Kan)
  • incubate over night at 37 °C

Transformation Efficiency 'Competent Cell Test Kit'

iGEM HQ

http://parts.igem.org/Help:Transformation_Efficiency_Kit

  1. spin down DNA (0.5, 5, 10, 20 & 50 pg/μL RFP Construct BBa_J04450, pSB1C3) from Competent Cell Test Kit (30 sec, 10.000 rpm)
  2. thaw competent cells on ice, label one 2 mL tube for each concentration + pre-chill
  3. 1 μL of DNA in each tube, respectively
  4. add 50 μL of competent cells to each tube, incubate for 30 min on ice
  5. pre-heat (42 °C), heat shock for 1 min
  6. incubate for 5 min on ice
  7. add 200 μL SOC media, incubate for 2 h at 37 °C
  8. pipet 20 μL on each plate (triplets), respectively
  9. incubate overnight at 37 °C
  10. count colonies, calculate transformation efficiency

Transformation

Zhang Gong

Cell type Purpose Cell amount DNA amount Colonies
DH5α Plasmid 50˜70 μL 0.1˜0.5 μL >10000
DH5α Ligation (Zhang's ligation protocol) 150 μL 10 μL 10˜500
DH5α Mutation 75˜150 μL 4˜5 μL 5˜100
BL21 Plasmid 75 μL 1˜2 μL 20˜100
Note: each tube of competent cell is 150 μL. For some purposes, if you have more than one sample to transform, you should split the cells in order to save work to make competent cells. Do not put the thawed competent cells back to -80 °C.

Procedure:

  1. Take competent cells from -80 °C. Thaw on ice (15˜30 min).
  2. Split cells, if necessary.
  3. Add DNA and mix.
  4. Put on ice 45 min˜2 h. (For DH5α plasmid preparation, 35 min is enough).
  5. Heat-shock: 42 °C 45 sec˜ 1 min. Then put back on ice for 2˜5 more minutes. Pre-warm 850 μ LB medium at the same time.
  6. Add pre-warmed LB medium into the competent cells. 37 °C 900 rpm 1 h.
  7. Centrifuge 4500 rpm 2 min to collect cells (if necessary). Put cells on appropriate plates or medium. 37 °C growing overnight.


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