Cloning

3.8.1. Ligation

Clonal PCR products are ligated into pGEM-T vector system I (Promega).

Ligations reactions are incubated at 4°C overnight.

3.8.2. Transformation

Transformation into JM109 competent Escherichia coli cells (Promega) follows:

1. Gently mix 5 | L of ligation product with 50 | L of JM109 cells and leave on ice for 20 min.

2. Heat shock cells at 42°C for exactly (use a timer!) 45 s and return to ice for 5 min.

4. Plate cells onto X-Gal/0.1 M Isopropyl P-D-thiogalactopyranoside, Amp100 Agar and leave overnight at 37°C.

The next day, 12 randomly picked bacterial colonies are each used to spike 2 mL Amp100 LB. Grow bacterial cultures at 37°C overnight.

3.8.3. Plasmid Purification

Plasmid is purified from the bacterial cultures using the QIAprep Miniprep kit (QIAGEN ).

1. Pellet cultures by centrifugation at 7000g for 1 min and resusupend in 250 |L of buffer P1.

2. Add 250 ||L of lysis buffer P2 and mix by inversion.

3. Neutralize the reaction within 5 min by the addition of 350 ||L of buffer N3. Leaving the alkaline solution for longer will hydrolyze the DNA (see manufacturer's instructions).

4. After centrifugation at 20,000g for 10 min, apply the supernatant to the column and leave for 1 min.

5. Centrifuge at 20,000g for 1 min

6. Wash once with buffer PB, followed by two washes with buffer PE.

7. Ensure a final centrifugation at 20,000g for 3 min before eluting DNA with 100 ||L of buffer EB

All plasmids are sequenced in both directions using T7 and SP6 forward and reverse primers. We aim to collect approx 10 clonally related sequences to confidently assess intraclonal variation in FL.

4. Notes

1. When working with RNA, gloves should be worn at all times and touching contaminated surfaces and equipment should be avoided; RNases can be reintroduced by contact with ungloved hands and equipment or with unfiltered air. Use sterile, RNase-free tubes and pipet tips and purchase reagents that are free of RNases. Separate reagents and plasticware used for RNA work from general-use reagents in the laboratory. Place all RNA samples on ice; RNA is susceptible to degradation when left at room temperature.

2. If no amplified products result from the first round of PCR, check cDNA integrity with P-actin primers if not previously performed. If, subsequently, (1) P-actin PCR fails, repeat cDNA synthesis, ensuring that positive and negative controls are run in parallel, and repeat using new cDNA synthesis reagents if necessary. In the event that the P-actin PCR fails again and assuming that cDNA synthesis reagents are viable, proceed to check the quality of the RNA and assess sample collection and storage. (2) If P-actin PCR successful, then the cDNA is viable. Repeat PCR using more cDNA template OR increase the number of PCR cycles OR lower primer annealing temperatures OR try alternative primer combinations OR use the above in combination.

3. If amplified products are weak, repeat PCR using more cDNA template OR increase the number of PCR cycles OR use in combination.

4. If multiple products are amplified from a single primer pair, excise, purify, and sequence each individual band that falls within the expected size range. Alternatively, repeat the PCR using standard (i.e., not "touchdown") cycling conditions and an increased annealing temperature.

5. If the sequencing of PCR product fails, check that the sequencing primer is correct. If not, repeat the sequencing reaction using more template DNA. If this coincides with a low yield of the original PCR product, disregard this product and attempt to increase the yield of product in a repeat PCR (refer to Subheading 4.2.) before resequencing.

6. If the sequencing of the PCR product appears clean but does not align to the VBase database, disregard this product. It is not Ig and most probably results from contamination for example from bacterial DNA.

7. If the sequence of the PCR product will (1) align to a V segment but not a J segment or (2) not align to a V or a J segment, examine the electropherogram for the pres ence of "double peaks" which indicate the existence of more than one sequence. Pay particular attention to the CDR3 region, from the end of V to constant, which may be polyclonal even if the V segment appears clonal. This PCR product is polyclonal and likely results from the amplification of V genes from normal B-cells, which exist within the tumor cell population.

8. If the cloning of the PCR product yields a low number of related sequences, purify more plasmids from the transformation plate for this PCR product AND/ OR clone out the second confirmatory PCR.

9. If cloning of the PCR product yields clonally related sequences but also a second group of sequences (>2) that share a clonally related CDR3 different to that already identified and you are confident that this is not the result of contamination and the tumor is indeed biclonal, purify more plasmids from the transformation plate for this PCR product and determine the dominant clone. We have observed dual light chain rearrangements in approx 8% of FL, whereas we have not found any tumors that proved to be biclonal for VH.

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