Clone antibody from Hybridoma
My protocol
[edit]
1. Extract of total RNA In general, 0.5 µg to 1.0 µg total RNA is sufficient for the amplification of specific Ig variable regions by 5’-RACE.
2. 1st strand synthesis using and MMLV reverse transcriptase
2490-mIgG2aHC-RT-R
(gagctcagcacccacaaaac)
gttttgtgggtgctgagctc
2 µg purified total RNA;
1 µg of appropriate constant region primer (2490-mIgG2aHC-RT-R)
5 µl 5× reaction buffer;
25 U RNasin Ribonuclease Inhibitor;
200 U M-MLV reverse transcriptase;
0.5 mM dNTP;
Nuclease-free water
Before adding the rest of the reaction mixture, prepare the RNA and corresponding
constant region primer in a separate tube, and heat to 70 °C for 10 min, to disrupt any RNA
secondary structure that might have formed. After heating, place the tube immediately on ice
to prevent secondary structure reforming. RNA can form very stable secondary structures that
can markedly reduce the efficacy of reverse transcription. Add the rest of the reverse
transcriptase mix to the tube, mix gently, spin down, and incubate at 42 °C for 60 min.
3. TdT tailing
Purify RT reaction by column.
Elute DNA with 50 ul H2O
Mix:
6.0 μl 10X TdT Buffer
6.0 μl 2.5 mM CoCl2 solution provided
50 μl 1st strand cDNA (~ 5.0 pmols DNA, i.e., 330 ng for 100 bp, 1 µg for 300 bp, 10 pmols DNA ends)*
1 μl 10 mM dATP
0.6 μl Terminal Transferase (20 units/μl)
- To determine approximate amount of DNA (ng/pmol), multiply the number of base pairs by 0.66. Example: 300 bp x 0.66 = 198 ng/pmol. For 5.0 pmols multiply by 5, resulting in 990 ng/5 pmol.
Incubate at 37°C for 30 minutes.
Cool on ice.
Terminal Deoxyribonucleotide Transferase (Promega, Madison, WI, USA);
dNTP (e.g. dATP) (Fermentas, Vilnius, Lithuania).
A homopolymeric tail is added to the 3’-end of the first-strand cDNA in a reaction that is
catalysed by terminal deoxynucleotidyl transferase (TdT) using only one of the possible four
dNTPs. Usually, 20-100 nucleotides are added to the DNA fragment in the tailing reaction,
and these represent a primer binding site for second-strand synthesis.
For addition of the poly(A) tail, prepare the following reaction mixture in a sterile
microcentrifuge tube on ice, in a total reaction volume of 20 µl:
5-10 µl purified first-strand cDNA (the optimal amount depends on the amount of RNA
used in the first-strand reaction and on the purification efficiency);
4 µl 5× reaction buffer;
150 pmol dATP (or any other dNTP);
30-40 U TdT;
Nuclease-free water.
Incubate for 1 h at 37 °C, and then for 10 min at 70 °C, to inactivate the enzyme. Place
the tube on ice. Isolate the poly(A)-tailed cDNA with a DNA Purification kit. TdT buffer is
not compatible with the PCR buffer system, therefore the tailed cDNA has to be purified
before PCR.
2. Synthesis of second-strand cDNA and PCR amplification of the Ig variable-region sequence
2494_IgG2a-HL-Const-R
constant
GGTCAAGGTGACTGGCTCAGGGAA
(ttccctgagccagtcaccttgacc )
2494_IgG2a-HL-Const-BamH1-F
actagCTCGAGcatatgggatccTTTTTTTTTTTTTTTTT
Finally, the Ig-coding variable region is amplified in a PCR reaction using two primers:
one complementary to the 5’-end (constant region) and the other complementary to the
homopolymeric tail at the 3’-end of first-strand cDNA. Phusion HF polymerase
is preferred for RACE, due to its lower error rate.
Prepare the following PCR reaction mixture, in a total reaction volume of 50 µl:
10 µl 5× reaction buffer;
0.2 µM constant region primer (e.g. constant region primer 2494_IgG2a-HL-Const-R GGTCAAGGTGACTGGCTCAGGGAA )
0.2 µM homopolymeric 2494_IgG2a-HL-Const-BamH1-F actagCTCGAGcatatgggatccTTTTTTTTTTTTTTTTT
0.2 µM dNTP mix;
2.5 U Phusion polymerase;
1-5 µl purified tailed first-strand cDNA;
Nuclease-free water.
Incubate in a thermal cycler preheated to 95 °C, using the following conditions:
Initial denaturation at 95 °C for 5 min, followed by 33 cycles of:
95 °C for 1 min;
55 °C for 1 min;
68 °C for 1 min.
Incubate at 68 °C for 10 min, then cool to 4 °C and store at -20 °C.
5. Clone into pMiniT vector
6. Sequencing
Protocol 2
[edit]based on this paper http://onlinelibrary.wiley.com/doi/10.1016/S0014-5793(99)00649-3/full
2.2 Primer design
Two databases of 491 mouse heavy chain and 356 mouse light chain signal sequences, expanded from the Kabat database ([32], http://immuno.bme.nwu.edu/), were used to design the PCR primers. Each signal sequence was assigned to a Vκ or a VH gene family [23,25], according to the related reference listed in the Kabat database. The signal sequences belonging to the same Ig gene family were aligned and the most conserved region was chosen to define consensus signal sequence primers as depicted on Tables 1 and 2. The antisense primer RevCκSalI (5′-CGACTA GTCGAC TGGTGGGAAGATGGATACAG-3′) contains the reverse complement of codons 113–120 of kappa light chain constant region. The following antisense oligonucleotides were designed to match the heavy chain constant regions: RevCγ1 SalI (5′-CGACAA GTCGAC TAGCCCTTGACCAGGCATCC-3′; matching codons 141–147 of Cγ1), RevCγ2a SalI (5′-CGACAA GTCGAC TAACCCTTGACCAGGCATCC-3′; matching codons 141–147 of Cγ2a), RevCγ2b SalI (5′-CGACTA GTCGAC CAGGGATCCAGAGTTCCAAG-3′; matching codons 156–166 of Cγ2b) and RevCγ3 SalI (5′-CGACTA TAGCCTTTGACAAGGCATCC-3′; matching codons 141–147 of Cγ3). These primers contained a SalI restriction site (underlined) for further cloning. The 5′-CG-3′ dinucleotide found in this restriction site is extremely rare in mammalian genomes [33] and the frequency of SalI recognition sites in variable domains of mouse antibodies is approximately 0.1–0.2%
Design PCR primers based on Table 1 and Table 2.
2.3 cDNA synthesis and PCR amplification of VH and Vκ genes from mouse hybridomas
Total RNA was extracted from 2×108 hybridoma cells using the TRIzol technique (Gibco BRL, Grand Island, NY, USA) as described by the manufacturer. 50 μg of total RNA was annealed at 65°C for 5 min with 10 pM of RevCκSalI primer for light chain cDNA synthesis or appropriate RevCγSalI primer for heavy chain cDNA synthesis in a 50 μl final volume of 1×first-strand cDNA synthesis buffer (Gibco BRL) containing 10 μM dNTPs (Gibco BRL) and 1 mM DTT (Gibco BRL). After annealing, the samples were cooled for 20 min at 20°C. 400 U of reverse transcriptase Superscript II (Gibco BRL) and 40 U of RNase inhibitor (Promega, Madison, WI, USA) were then added and the reaction was incubated for 1 h at 42°C. Reverse transcriptase activity was inactivated by incubation at 95°C during 5 min and cDNA was kept at −20°C until use. PCR amplification was carried out in a final volume of 20 μl containing 1 μl of cDNA synthesis reaction, 10 μM dNTPs, 2 μl of 10×PCR buffer (New England Biolabs, Beverly, MA, USA). Fourteen VH and 18 Vκ PCR reactions were set up using each group of family-specific 5′ primers (Tables 1 and 2) and the appropriate 3′-oligonucleotide probe matching the light or heavy chain constant region. Two mixtures, containing all VH or Vκ 5′ primers, were also set up. No PCR reaction was performed with primer specific for the VH13 (3609N) gene family since the only reported member assigned to this family is the non-functional allele of PC3609 [35]. The amount of each primer used was initially 10 pM for non-degenerated oligonucleotide and was increased two- to eight-fold depending on the degeneracy. The reaction mixtures were heated to 94°C for 10 min, then 2 U Vent DNA polymerase (New England Biolabs) was added and 30 cycles of amplification were carried out for 1 min at 94°C, 1 min at 55°C and 2 min at 72°C. After a 10 min extension at 72°C, the PCR products were fractionated through a 1.5% agarose gel (Gibco) and stained with ethidium bromide. Sets of 5′ primers and 3′ primers leading to a 390 bp product for Vκ amplification and a 450 bp product for VH amplification were selected from this family-specific PCR screening. Five replicates using the same selected primers were subjected to a new PCR as described above. The PCR-amplified DNA products were gel purified on a 1.5%, low melting temperature agarose gel (Gibco).
IgG2a
RT primers:
RevCγ2a SalI for IgG2A Heavy chain (5′-CGACAA GTCGAC TAACCCTTGACCAGGCATCC-3′; matching codons 141–147 of Cγ2a),
RevCκSalI for IgG2A light chain (5′-CGACTA GTCGAC TGGTGGGAAGATGGATACAG-3′)
PCR Primers IgG2A:
http://www.protocol-online.org/forums/uploads/monthly_12_2010/msg-6759-075982100%201291964115.ipb
Mouse heavy chain constant region primers
5 -gga aga tct CTT GAC CAG GCA TCC TAG AGT CA-3
Mouse Light kappa chain constant region primer
3' Kc: 5 -ggt gca tgc GGA TAC AGT TGG TGC AGC ATC-3
Mouse kappa chain FR1 region universal degenerate primer
L Chain
5' Mk is used as the 5' primer for the VL amplification
Sense
5' Mk: 5 -gg gag ctc GAY ATT GTG MTS ACM CAR WCT MCA-3
Antisense
3' Kc: 5 -ggt gca tgc GGA TAC AGT TGG TGC AGC ATC-3
2486-TP4-mIgG2a-LC-VR-Xho1-R
(GCAGATGCTGCACCAACTGTATCC ctcgag atagca )
tgctatctcgagGGATACAGTTGGTGCAGCATCTGC
2486-TP4-mIgG2a-LC-VR-EcoR1-F
tagaca gaattc GAY ATT GTG MTS ACM CAR WCT MCA
H Chain
An equimolar mixture of 5' MH1 and 5' MH2 as the 5' VH primer
Sense
5' MH1: 5 -ctt ccg gaa ttc SAR GTN MAG CTG SAG SAG TC-3
5' MH2: 5 -ctt ccg gaa ttc SAR GTN MAG CTG SAG SAG TCW GG-3
2485-TP4-EcoR1-F1 5 -ctt ccg gaa ttc SAR GTN MAG CTG SAG SAG TC-3
2485-TP4-EcoR1-F2 5 -ctt ccg gaa ttc SAR GTN MAG CTG SAG SAG TCW GG-3
Antisense
IgG2A: 5 -gga aga tct CTT GAC CAG GCA TCC TAG AGT CA-3
2485-TP4-Nhe1-Xho1-R
gtgcta ctcgag GCTAGC cttgaccaggcatcctagagtcac
(gtgactctaggatgcctggtcaag GCTAGC ctcgag tagcac)