IgG sequencing of rat hybridoma

TS pF is ordered as RNA oligo; the remainder as DNA oligo

universal TS pF AAGCAGTGGTATCAACGCAGAGTACATrGrGrG

ISPCR pF aagcagtggtatcaacgcagag

rat IGHG RT pR GGACAGGGCTCCAGAGTTCC

rat IGHG PCR pR GACTGGCTCAGGGAAATAGCC

rat IGKC RT pR CTGATCAGTAACACTGTCCAGGAC

rat IGKC PCR pR CACTGATGTCTCTGGGATAGAAGTTG

rat IGLC1 RT pR GGGAGATAGGTGCACCATTTGC

rat IGLC1 PCR pR GGCCACTTCCACATCACTCG

rat IGLC2 RT pR TCCACACCCTGAGTGATAGGG

rat IGLC2 PCR pR CTTCCAGACCACTGTCATAACACC

RNA extraction: Extract RNA from the hybridoma sample using RNeasy total RNA kit, according to the manufacturer’s instructions.

Reverse Transcription: On ice, prepare the 1^st reaction mix in PCR tubes for gamma chain and kappa chain (or lambda chain) cDNA synthesis according to the following recipe:

Gamma

A	B
Component	Volume per rxn
IGHG RT pR (10 uM)	1 uL
dNTP (10 mM)	1 uL
RNA sample (50 ng/uL)	2 uL
Total volume	4 uL

Kappa

A	B
Component	Volume per rxn
IGKC RT pR (10 uM)	1 uL
dNTP (10 mM)	1 uL
RNA sample (50 ng/uL)	2 uL
Total volume	4 uL

Lambda (include later if Kappa does not amplify)

A	B
Component	Volume per rxn
IGLC1 (or C2) RT pR (10 uM)	1 uL
dNTP (10 mM)	1 uL
RNA sample (50 ng/uL)	2 uL
Total volume	4 uL

On ice, prepare a 2^nd mastermix in Eppendorf tubes. This recipe if for one reaction regardless of chain type.  Scale up for the number of samples as needed.

2nd reaction mix ^nd reaction mix

A	B
Component	Volume per rxn
5x SMARTScribe buffer	2 uL
DTT (20 mM)	1 uL
Universal TS pR (100 uM)	0.3 uL
H2O	1.70 uL
Total volume	5.00 uL

After preparing the 2^nd mastermix, incubate each of the 1^st reaction mixes in a thermocycler for  3 minutes at 72°C.

While the incubation reaction is proceeding, add the following to the 2^nd reaction mix:

A	B
Component	Vol. per rxn
RNAse inhibitor (40 U/uL)	0.50 uL
SMARTScribe Rev. Transcriptase (100 U/uL)	0.50 uL
Total volume	1.00 uL

Once the incubation of the 1^st reaction mix (from step 3.5) finishes, add 6 uL of the 2^nd reaction mix to each tube of the 1^st reaction mix.

With the 2 reaction mixes now combined, incubate each according to the following conditions:

A	B	C
Temperature	Time	Cycles
42°C	60 min	1
70°C	5 min	1
4°C	hold	-

Proceed to PCR amplification step immediately after incubation has finished (once samples reach 4°C hold step).

PCR amplification: Prepare mastermix for PCR reaction according to following 2-step recipe:

Add the following components to each PCR tube.

Gamma

A	B
Component	Volume per rxn
Platinum SuperFi II PCR MM	25 uL
ISPCR pF (10 uM)	2.5 uL
IGHG PCR pR (10 uM)	2.5 uL
cDNA from RT step (5-100ng)	3 uL
Water, nuclease-free	17 uL
Total volume	50 uL

Kappa

A	B
Component	Volume per rxn
Platinum SuperFi II PCR MM	25 uL
ISPCR pF (10 uM)	2.5 uL
IGK PCR pR (10 uM)	2.5 uL
cDNA from RT step (5-100ng)	3 uL
Water, nuclease-free	17 uL
Total volume	50 uL

Lambda (if Kappa doesn’t amplify)

A	B
Component	Volume per rxn
Platinum SuperFi II PCR MM	25 uL
ISPCR pF (10 uM)	2.5 uL
IGLC1 (or C2) PCR pR (10 uM)	2.5 uL
cDNA from RT step (5-100ng)	3 uL
Water, nuclease-free	17 uL
Total volume	50 uL

Cap each tube, then mix and briefly centrifuge the PCR tubes.

Place PCR tubes in thermocycler, and run according to the following conditions:

Gamma

A	B	C
Temperature	Time	Cycles
98°C	30 sec	1
98°C	15 sec	35
63°C	30 sec
72°C	25 sec
72°C	5 min	1
4°C	hold	-

Kappa/Lambda

A	B	C
Temperature	Time	Cycles
98°C	30 sec	1
98°C	15 sec	35
56°C	30 sec
72°C	25 sec
72°C	5 min	1
4°C	hold	-

Verify the gel bands

After PCR reaction completes, set up 1% agarose gel according to the following conditions:

In a flask, add the following components:

0.5 g – Agarose powder

50 mL – 1X TAE buffer

Put flask, with added reagents, in microwave and heat up until all the powder is fully dissolved (about 1 minute).

After microwaving is finished, remove flask with hot pad; add 5 uL of Sybrsafe to flask, swirl to mix. Pour into a tray with appropriate plastic comb, preferably one with wells that accommodate 20uL volumes.

Prepare a fraction of each PCR products to verify the band size on the gel. Aliquot 5 uL from each PCR tube into a new PCR strip, and add 1 uL of DNA Loading Dye to each sample.

Load the gel and run it for 25-30 minutes at 110 V.  Modify these settings if needed.

Remove gel, image, and save an image copy for the records.

Note:  There should be a gamma chain, and either a kappa or lambda chain.

Amplified rat antibody products: 550-600 bp.

A significant majority of mouse antibody light chains will be kappa; if kappa not present, repeat with IGLC1 and IGLC2 samples.

PCR cleanup

PCR samples that produce a band of the expected size can then be finalized using a PCR purification kit (e.g. Invitrogen's PureLink™ PCR Purification Kit #K310001), and eluted in 25 uL volume. Verify cDNA concentration by nanodrop.

Sanger sequencing should be performed (e.g. GeneWiz or Genomic Core).

Analyze the antibody nucleotide sequence

Open the website: https://www.imgt.org/IMGT_vquest/vquest

Copy and paste sequence into the IMGT webpage, in the section “sequence submission”.

Select “Rattus norvergicus” for species, and “IGH” as receptor type or locus.

Click on "Start".