1

Content
RT-PCR ...................................................................................................................................... 2
Nested PCR................................................................................................................................ 2
Agarose Gels .............................................................................................................................. 4
Loading Dye (1M sucrose, 0.02 cresol red) ............................................................................... 4
DNA Ladder (Marker) ................................................................................................................. 4
Load PCR product on Gel .......................................................................................................... 4
Purification of PCR product PCR Kit ....................................................................................... 5
Sequencing................................................................................................................................. 5
Digestion of PCR product........................................................................................................... 6
Vector Digestion ......................................................................................................................... 7
Ligation ....................................................................................................................................... 8
Transformation ........................................................................................................................... 9
Agar Plates: ................................................................................................................................ 9
LB Medium:................................................................................................................................. 9
Insert Check PCR ......................................................................................................................10
Inoculation .................................................................................................................................11
Glycerol Stock: ..........................................................................................................................11
Using Glycerol Stock: ................................................................................................................11
Miniprep/Midiprep/Maxiprep ......................................................................................................12
PEI transfection of 293F cells ...................................................................................................13
Cell Harvest ...............................................................................................................................13
Antibody purification ..................................................................................................................14
Glycine for mAb ........................................................................................................................14



2

RT-PCR
1. Prepare the two mix in eppendorf tubes:
RHP
mix
reagent concentration 230x
water - 1081,00
RHP [300 ng/l] 230,00
NP-40 [10%] 230,00
RNAsin [40 U/l] 69,00
total 1610,00

RT mix reagent concentration 230x
water - 920,00
First strand
buffer
[5x] 1380,00
DTT [100 mM] 460,00
dNTPs [25 mM each] 230,00
RNAsin [40 U/ul] 92,00
SuperScript III [200 U/l] 138,00
total 3220,00
1. First add 5l RHP mix to each well of the 96 well PCR plate, then put it on the
heating plate for 1 minute at 68C.
2. Add 10l of the RT mix to each well and then start the PCR machine.
Nested PCR

1PCR reagent concentration 1x 210x
water - 32,16 6753,60
buffer [10x] 4,00 840,00
primer A [50 M] 0,13 27,30
primer B [50 M] 0,13 27,30
dNTPs [25 mM each] 0,40 84,00
HotStar Taq [5 U/l] 0,18 37,80

subtotal
mix
37,00 7770,00

template 3,00
total 40,00

Primers for Gamma: L-VHmix, CgCH1 red plates
Primers for lLambda: LV mix, C1 green plates
Primers for Kappa: LV mix, CK1 yellow plates
3

Add 37l of the master mix to the right coloured 96 well plate. Then add 3l/well of
the cDNA to the plate.
Program the PCR machine:
Gamma and Kappa: Lambda:
Temperature Time
95C 15 min
94C 0.30min
58C 0.30min 50 cycles
72C 0.55min
72C 10min
4C

2PCR reagent concentration 1x 210x
water - 31,66 6648,60
buffer [10x] 4,00 840,00
primer A [50 M] 0,13 27,30
primer B [50 M] 0,13 27,30
dNTPs [25 mM each] 0,40 84,00
HotStar Taq [5 U/l] 0,18 37,80

subtotal
mix
36,50 7665,00

template 3,50
total 40,00

Primers for Gamma: Agel VHmix, SallJH mix red plates
Primers for lambda: AgelVmix, XholC1 green plates
Primers for Kappa: AgelVKmix, BsIWI JKmix yellow plates
Add 36l to each well of a new and right coloured 96well plate. Then transfer
4l/well of the first PCR to the 2
nd
PCR plate.
Program the PCR machine:
Gamma and Kappa: Lambda:
Temperature Time Temperature Time
95C 15 min 95C 15 min
94C 0.30min 94C 0.30min
58C 0.30min 50 cycles 58C 0.30min 50 cycles
72C 0.55min 72C 0.55min
72C 10min 72C 10min
4C 4C

Temperature Time
95C 15 min
94C 0.30min
58C 0.30min 50 cycles
72C 0.55min
72C 10min
4C
4

Agarose Gels
1. Add 4 g Agarose Powder to 200 ml
of 1 x TBE
2. Use the program 5 in the microwave heat it until the agarose powder is
dissolved
3. Assemble the apparatus.
4. Add 8 l of Gel Red to the Gel
5. Pour into apparatus when you can touch the flask


Loading Dye (1M sucrose, 0.02 cresol red)
1. 17g sucrose dissolved to a total volume of 49 ml water
2. Add 1000 l 1% cresol red (0.05g/ 50 ml)


DNA Ladder (Marker)
1. Mix 50l of DNA ladder (freezer) with 300l blue dye (upper drawer in the
fridge door)
2. Add 150l TBE (or more, depends on how concentrated your marker is)

Load PCR product on Gel
Mix 10l of loading dye with 10l of 2
nd
PCR product -> Load 15l into each
pocket, and run the Gel at constant 180V


















5


Purification of PCR product PCR Kit
1. Label wells on PCR plates and centrifuge it down
2. Pull a whole into the labelled wells and transfer the content of the well into the
tubes (around 30l)
3. Add pH indicator to PB Buffer (50ml Buffer -200l pH indicator)
4. Add 150l PB Buffer to each tube and mix if the colour is orange or violet
add 2-5l Sodium acetate to each tube
5. Put spin columns to vacuum device and transfer tube content into the spin
column
6. Apply pressure until liquid is gone (between 20 and 30mbar)
7. Add 800l of washing buffer to each column -> pressure around 50mbar
8. Apply vacuum at least for 10 minutes
9. Centrifuge tubes at 13000rpm for 2 minutes
10. Transfer filter of the spin column to new tubes
11. Add 100 l Elution Buffer and wait for 2 minutes
12. Spin the tubes down by 13000rpm for 2 minutes

Sequencing
Primers:
Chain PCR product Plasmid
Gamma 5 AgelVH mix (VH1/5,VH3,VH4,VH3-23,VH4-34) IgG Internal
Lambda 3 XholCL CL1
Kappa 5 panVK CK1

1. Add water and PCR product/ Plasmid to 96 well plate (it should be at least half
full)
2. Prepare Excel Sheet with the position, names, chain of the specific PCR
products
3. Dilute the primers from 50 pmol/l to 10 pmol/l. There should be enough to
put 2l of the primers into each well. (Volume should be over 50l)
Calculation: number of wells x 2 = total volume
Total volume /5 = amount of primer
Total volume amount of primer = amount of water
6

Digestion of PCR product
1. For Gamma and Lambda PCR product
Prepared Mix- Content for one well/tube:
Water 14 l
Smart Buffer 5 l Vortex!

Endonuclease 0.5 l each Spin down the
enzymes Endonuclease
- Sall and AgeI for Gamma
- Xhol and and AgeI for Lambda


Add 20 l of the mix to each well/tube
After Centrifuge down and mixing the sample add 30 l of it.
Incubate for 2h at 37C in the heating block
If you use the other buffer you dont have to add 0.5 l BSA!
2. Prepare first mix for Kappa (one well content)
Water 13.5 l
Buffer 5 l Vortex!
BSA 0.5 l
Endonuclease Agel 0.5 l Spin down!

Add 20l of the mix to each well/tube
After centrifuge down and mixing the sample add 30l of it.
Incubate for 2h at 37C in the heating block

3. Second Mix for Kappa PCR product:
Water 13.5 l
Buffer 5 l Vortex!
BSA 0.5 l
Endonuclease Agel 0.5 l Spin down!

Spin down the tube/well
Add 10l of each mix to each well/tube (vortex gently)
Incubate for 2h at 55C in the heating block

7

Perform PCR clean-up for the digested product as described above with the PCR
product!
Vector Digestion
1. Prepare 1
st
Digest Mix:
Water 37 l
Buffer 6 l Vortex!
BSA 0.6 l
Endonuclease Agel 6 l Spin down!
Vector 10

2. Incubate for 2 hour at Room Temperature
3. Add 3l Agel
4. Incubate for 2-3 hour at 37C
5. Prepare 2
nd
Digest Mix
Gamma Lambda Kappa
Water 17 l 17 l 17 l
Buffer 10 l 10 l 10 l
Endonuclease SalI 10 l Xhol 10 l BsiWI 10 l

6. Incubate Gamma and Lambda for 2 hours at 37C and Kappa 2 hours at
55C
7. Add 3l of Sall/Xhol/BsiWI
8. Incubate overnight at 37C (Gamma,Lambda) or 55C (Kappa)
9. Purify with the PCR purification Kit described above
10. Elute in 50l Elution Buffer









8

Ligation
Set up per well of a PCR plate:
Water 2 l
Buffer 25 l
Vector 1 l
Digested PCR
product
20 l
Ligase 2 l

1. Prepare a mix with Vector, Water and Vector and add 28 l of it to each well/tube.
(on ice)
2. Add 20 l of DNA to each tube/well
3. Add 2 l of Ligase to each tube/well
4. Incubate 15 minutes at 25C in the heating block
5. Put the tubes back on ice
PS. The quantity of the vector should be between 40 ng/ul and 100 ng/ul!!!





























9



Transformation
1. Throw Bacteria (calculate before how much you need; 50l per reaction around
500 ml in one tube)
2. Transfer 80 l of bacteria to a tube or well
3. Add 5 l of ligation product to the tube/well
4. Mix gently and incubate 30 min on ice
5. Put the tubes/plate on 42C for 45 seconds and back on ice after that
6. Add 200 l LB medium (without ampicillin) to the tube or on a deep well plate
and transfer the bacteria to the deep well plate
7. Incubate for 30 minutes at 37C
8. Take the plates out of the cold room and label them
9. Add around 150 l of the bacteria to the Agar plates
10. Let them dry and incubate them over night at 37C
Agar Plates:
1. Take a 1000ml flask
2. Same or one day before you can already weight out 14 g of LB-powder and
10.5 g Agar (Bact). If there is no Agar (bact) in the drawer you can find more
in the chemical room at level 4.
3. Before 10 o clock add 700 ml of distilled water and shake. Put aluminium foil
on top of the lid and fix it with autoclaving tape.
4. Bring it to the autoclaving room upper drawer its marked with in.
5. Around 13:00 you can pick up the Medium. If you have more than one bottle
but the other into the water bath.
6. Add 700 l ampicillin to the 700ml medium
7. Pour liquid into plates
LB Medium:
1. 20g of LB powder and add 1000 ml distilled water
Autoclaving
2. If you want to have LB with Ampicillin add 100 l to 100 ml or 1000l to
1000ml

10


Insert Check PCR
PCR Mix prepared as following: (for 200 wells)
Water 3300l
5x Buffer 1000l
dNTPs (1.25mmol -> 20 time
dilution)
500l
Primers Each
40l
MgCl2 400l
Tag 50l

11. 25l to each well of the PCR plates
Take a 10l tip and take the colony up. Spread the colony on a backup-LB plate. Be
careful! Dont take up the agar, just touch the surface. Put the tip into the Plate and
mix gently.
Incubate the Backup-plate for 3 hours at 37C
Program Mastercycler:
Temperature Time
94C 5 min
94C 0.30min
58C 0.30min 30 cycles
72C 0.55min
72C 10min
4C

Expected size on the gel:
Gamma: 650 bp Lambda: 590 bp Kappa: 700 bp









11


Inoculation
1. Add 8 ml LB medium with ampicillin into a 15ml tube. (100g Ampicllin in 1
2. Take up the positive colony from the backup-plate and throw it into the tube.
3. Incubate over night at 37C with shaking (200rpm)
Glycerol Stock:
1. Prepare a glycerol LB stock solution of 50% glycerol and 50% LB medium
2. Add 200 l of the inoculate to 150 l of the glycerol-LB medium
3. Store it at -20C
Using Glycerol Stock:
1. Add 10-20 l of the glycerol stock to 200 l of LB medium
2. Incubate for at least 4 hours at 37C in the heating block
3. After 4 hours you can increase the volume


















12

Miniprep/Midiprep/Maxiprep































13

PEI transfection of 293F cells
1. The day before transfection, pass 293F cells to 0.6-0.7 x 10
6
cells/ml.
2. Mix 38 ug of total plasmid DNA, half with heavy chain (19 ug) and half with
light chain (19 ug).
3. Dilute PEI-MAX (PEI-40, Polysciences) to have 120 ug in 1.2 ml of OPTI-
PRO SFM for each transfection. For e.g. 48 transfections, mix 70 ml OPTI-
PRO SFM with 7 mg PEI-MAX.
4. The day of transfection, adjust cells to a concentration of 1 x 10
6
cells/ml in
Freestyle Medium. Add 30 ml of the media into a 125 ml flask. Put the flasks
back into the incubator.
5. Add 1.2 ml of PEI-MAX in OPTI-PRO SFM to labelled tubes. Add DNA
plasmid mix to the tube and vortex for 5 seconds. Incubate at room
temperature for 10 minutes.
6. Add DNA plasmid mix drop-wise to the 30 ml cell suspension.
7. Incubate at 37C in shaking incubator for 7 days.

Cell Harvest
1. Swirling the flask then pouring the contents into a 50 ml Falcon tube.
Following steps are not performed in tissue culture conditions.
2. Centrifuge tubes in a floor-standing centrifuge at 2000 rpm for 10 minutes
3. Slowly decant the supernatant by slowly pouring into a new 50 ml tube. The
cell pellet should not be disrupted.


















14

Antibody purification
1. Vortex Protein G Sepharose 4 Fast Flow beads (GE Healthcare) and shake
bottle vigorously. Add enough beads for 100 ul per sample to a 50 ml tube
containing PBS.
2. Wash beads by centrifugation at 2000 rpm for 10 mins with a brake of 2.
Take off supernatant using a pipette, then centri fuge again at 2000 rpm for
10 mins with low brake. Resuspend beads in PBS to achieve a total volume
of 400 ul per sample.
3. Cut off the end of a 1 ml pipette tip and add 400 ul of beads into the tube
containing the supernatant.
4. Attach the tubes containing 50 ml cell culture supernatant and 100 ul Protein
G beads to a rotor in the cold room using tape and incubate overnight (at least
14 hours) at 4C.
5. Centrifuge supernatants at 2000 rpm for 10 min with brake.
6. In the meantime, prepare 4 eppendorf tubes per sample containing 50 ul 1M
Tris (pH 8.0).
7. Remove supernatant using 50 ml pipette set at g without disrupting pellet and
discard supernatant. Leave 1ml of supernatant at the bottom with the
beads.
8. Add 2 ml of PBS to each chromatography column.
9. Resuspend the beads in the remaining liquid, and transferred to a
chromatography spin column.
10. Add 2ml PBS to bead-containing tubes, then transfer any remaining beads
stuck to the sides to the column.
11. Wash three times with 2 ml PBS.
12. Transfer columns to 1.5 ml eppendorf tube, blotting the bottom of the column
to get rid of excess flow through droplets.
13. Add 500 ul of filtered 0.1M glycine (pH 3.0) to the column to elute the
antibodies in fraction 1.
14. Transfer the column to a second, third then fourth eppendorf tube to elute
Fractions 2, 3 and 4 using 500 ul of glycine each time.

Glycine for mAb (0.1M; pH==3)
1. 3.75g glycine in 500ml MQ water
15

2. HCL for pH adjustment